xrn1  (New England Biolabs)


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    Name:
    XRN 1
    Description:
    XRN 1 100 units
    Catalog Number:
    M0338L
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    358
    Category:
    Ribonucleases RNase
    Size:
    100 units
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    Structured Review

    New England Biolabs xrn1
    XRN 1
    XRN 1 100 units
    https://www.bioz.com/result/xrn1/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xrn1 - by Bioz Stars, 2021-05
    99/100 stars

    Images

    1) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    2) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Determine the region of 3’ UTR involved in the accumulation of sfRNA constructed in the context of a full-length infectious JEV clone. (A) Predicted secondary structures within the 585-nt 3’ UTR of JEV genome. Nucleotides are numbered from the first base of the genome. The deletion or base substitution regions made in this study are marked or color-coded. (B) Schematic diagram of the 3’ UTR mutants and the results of Northern hybridization for detecting the genome and sfRNA are summarized on the right. (C) BHK-21 cells were infected with recombinant mutant viruses at an MOI of 0.1. RNAs were extracted at 48 h post-infection and Northern hybridization was done as described in Fig 1A . (D) HEK293T cells (WT) or XRN1-knockdown cells (KD) were infected with a PK disrupted mutant (PK1”), and a PK compensatory changed mutant (PK1’1”) viruses at an MOI of 1. Cytoplasmic RNAs were extracted at 60 h post-infection and analyzed by Northern blot. (E) Plaque assays of WT and all mutant viruses were done on BHK-21 cells. Cells were fixed and stained with naphthol blue–black dye at 4 days post-infection. (F) Viral growth kinetics of the WT infectious clone and the recombinant mutant viruses. BHK-21 cells were infected at an MOI of 0.1, and the supernatant fluid of the infected cells was sampled at the indicated times post infection. Titers were determined by plaque assays on BHK-21 cells.
    Figure Legend Snippet: Determine the region of 3’ UTR involved in the accumulation of sfRNA constructed in the context of a full-length infectious JEV clone. (A) Predicted secondary structures within the 585-nt 3’ UTR of JEV genome. Nucleotides are numbered from the first base of the genome. The deletion or base substitution regions made in this study are marked or color-coded. (B) Schematic diagram of the 3’ UTR mutants and the results of Northern hybridization for detecting the genome and sfRNA are summarized on the right. (C) BHK-21 cells were infected with recombinant mutant viruses at an MOI of 0.1. RNAs were extracted at 48 h post-infection and Northern hybridization was done as described in Fig 1A . (D) HEK293T cells (WT) or XRN1-knockdown cells (KD) were infected with a PK disrupted mutant (PK1”), and a PK compensatory changed mutant (PK1’1”) viruses at an MOI of 1. Cytoplasmic RNAs were extracted at 60 h post-infection and analyzed by Northern blot. (E) Plaque assays of WT and all mutant viruses were done on BHK-21 cells. Cells were fixed and stained with naphthol blue–black dye at 4 days post-infection. (F) Viral growth kinetics of the WT infectious clone and the recombinant mutant viruses. BHK-21 cells were infected at an MOI of 0.1, and the supernatant fluid of the infected cells was sampled at the indicated times post infection. Titers were determined by plaque assays on BHK-21 cells.

    Techniques Used: Construct, Northern Blot, Hybridization, Infection, Recombinant, Mutagenesis, Staining

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

    3) Product Images from "Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia"

    Article Title: Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia

    Journal: Viruses

    doi: 10.3390/v12091017

    Xrn1 degradation assay and determination of Xrn1 stop point. ( a ) Schematic diagram of the BJV and BJLV 3′-UTR xrRNA degradation assay. xrRNA structures (shown in the box) were challenged with Xrn1 enzyme. (1) BJV 3′-UTR xrRNA (2) BJLV 3′-UTR xrRNA (3) BJV 3′-UTR xrRNA ΔHP. ( b ) In vitro Xrn1 degradation assay against xrRNA structure of BJV and BJLV, and the hairpin structure of BJLV. Data show a representative result of two independent experiments. Examination of BJV ( c , e ) and BJLV ( d , f ) Xrn1 stop points, which we could unambiguously locate directly at upstream of the xrRNA structures. Red arrow: Stop point.
    Figure Legend Snippet: Xrn1 degradation assay and determination of Xrn1 stop point. ( a ) Schematic diagram of the BJV and BJLV 3′-UTR xrRNA degradation assay. xrRNA structures (shown in the box) were challenged with Xrn1 enzyme. (1) BJV 3′-UTR xrRNA (2) BJLV 3′-UTR xrRNA (3) BJV 3′-UTR xrRNA ΔHP. ( b ) In vitro Xrn1 degradation assay against xrRNA structure of BJV and BJLV, and the hairpin structure of BJLV. Data show a representative result of two independent experiments. Examination of BJV ( c , e ) and BJLV ( d , f ) Xrn1 stop points, which we could unambiguously locate directly at upstream of the xrRNA structures. Red arrow: Stop point.

    Techniques Used: Degradation Assay, In Vitro

    Diagrams of xrRNA structure. xrRNA structure of ( a ) BJV and ( b ) BJLV. Arrows indicate the three-way junction structure and arrowheads indicate Xrn1 stop point; PK: pseudoknot.
    Figure Legend Snippet: Diagrams of xrRNA structure. xrRNA structure of ( a ) BJV and ( b ) BJLV. Arrows indicate the three-way junction structure and arrowheads indicate Xrn1 stop point; PK: pseudoknot.

    Techniques Used:

    4) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Northern Blot, Produced, Staining

    A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of
    Figure Legend Snippet: A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of

    Techniques Used: Incubation, Purification, Recombinant, Activity Assay

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.
    Figure Legend Snippet: Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.

    Techniques Used: Sequencing, Northern Blot, Produced, Expressing, Plasmid Preparation, Staining

    5) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

    6) Product Images from "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells"

    Article Title: The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

    Journal: bioRxiv

    doi: 10.1101/852434

    Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .
    Figure Legend Snippet: Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .

    Techniques Used: Recombinant, In Vitro, Produced, Purification, Polyacrylamide Gel Electrophoresis

    7) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    8) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    9) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Northern Blot, Produced, Staining

    A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of
    Figure Legend Snippet: A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of

    Techniques Used: Incubation, Purification, Recombinant, Activity Assay

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.
    Figure Legend Snippet: Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.

    Techniques Used: Sequencing, Northern Blot, Produced, Expressing, Plasmid Preparation, Staining

    10) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Northern Blot, Produced, Staining

    A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of
    Figure Legend Snippet: A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of

    Techniques Used: Incubation, Purification, Recombinant, Activity Assay

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.
    Figure Legend Snippet: Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.

    Techniques Used: Sequencing, Northern Blot, Produced, Expressing, Plasmid Preparation, Staining

    11) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    12) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

    13) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

    14) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    15) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

    16) Product Images from "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells"

    Article Title: The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

    Journal: bioRxiv

    doi: 10.1101/852434

    Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .
    Figure Legend Snippet: Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .

    Techniques Used: Recombinant, In Vitro, Produced, Purification, Polyacrylamide Gel Electrophoresis

    17) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Northern Blot, Produced, Staining

    A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of
    Figure Legend Snippet: A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of

    Techniques Used: Incubation, Purification, Recombinant, Activity Assay

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.
    Figure Legend Snippet: Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.

    Techniques Used: Sequencing, Northern Blot, Produced, Expressing, Plasmid Preparation, Staining

    18) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Northern Blot, Produced, Staining

    A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of
    Figure Legend Snippet: A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of

    Techniques Used: Incubation, Purification, Recombinant, Activity Assay

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.
    Figure Legend Snippet: Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.

    Techniques Used: Sequencing, Northern Blot, Produced, Expressing, Plasmid Preparation, Staining

    19) Product Images from "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells"

    Article Title: The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

    Journal: bioRxiv

    doi: 10.1101/852434

    Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .
    Figure Legend Snippet: Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .

    Techniques Used: Recombinant, In Vitro, Produced, Purification, Polyacrylamide Gel Electrophoresis

    20) Product Images from "The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2"

    Article Title: The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2

    Journal: The EMBO Journal

    doi: 10.15252/embj.201488645

    SUMM2 is required for the constitutive defense phenotype of pat1 mutants A Elevated PR gene expression in pat1 mutants is suppressed by summ2-8 . Four-week-old plants were used for RNA extraction, followed by qRT-PCR. Fold-change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col-0, normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3). B pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2-8 . Bacteria were syringe-infiltrated and samples taken 3 days post-infiltration. Data are log 10 -transformed colony-forming units/cm 2 leaf tissue (cfu/cm 2 ). Standard error of the mean is indicated by errors bars ( n = 4). C 14-day-old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT-qPCR. Data were normalized to ACT2 , and transcript levels were compared between XRN1 and mock-treated RNA for each genotype. Standard error of the mean is indicated by error bars ( n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test. D Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col-0 and mkk1/1-summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.
    Figure Legend Snippet: SUMM2 is required for the constitutive defense phenotype of pat1 mutants A Elevated PR gene expression in pat1 mutants is suppressed by summ2-8 . Four-week-old plants were used for RNA extraction, followed by qRT-PCR. Fold-change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col-0, normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3). B pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2-8 . Bacteria were syringe-infiltrated and samples taken 3 days post-infiltration. Data are log 10 -transformed colony-forming units/cm 2 leaf tissue (cfu/cm 2 ). Standard error of the mean is indicated by errors bars ( n = 4). C 14-day-old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT-qPCR. Data were normalized to ACT2 , and transcript levels were compared between XRN1 and mock-treated RNA for each genotype. Standard error of the mean is indicated by error bars ( n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test. D Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col-0 and mkk1/1-summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.

    Techniques Used: Expressing, RNA Extraction, Quantitative RT-PCR, Transformation Assay, Mass Spectrometry

    21) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

    22) Product Images from "Cap homeostasis is independent of poly(A) tail length"

    Article Title: Cap homeostasis is independent of poly(A) tail length

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1460

    Appearance of uncapped forms of cytoplasmic capping targets in non-translating mRNPs. ( A ) Cytoplasmic extracts from uninduced and 12 h K294A-expressing cells were separated on linear sucrose gradients and pooled into mRNP and polysome fractions. ( B ) RNA recovered from the pooled mRNP was spiked with uncapped luciferase RNA as a control for Xrn1 digestion, and capped β-globin RNA as a control for sample recovery. Half of each sample was treated with Xrn1 to degrade uncapped RNAs ( 12 ) and the recovered RNA was assayed by RT-qPCR using primers located near the 5′ ends of luciferase, β-globin, β-actin, RPLP0,VDAC3, ILF2, ZNF207 and MAPK1 mRNA. Results were calculated as in ( 12 ) and are shown as the change in Xrn1 sensitivity of unaffected (β-actin, RPLP0) and target (VDAC3, ILF2, ZNF207 and MAPK1) mRNAs as a function of K294A expression.
    Figure Legend Snippet: Appearance of uncapped forms of cytoplasmic capping targets in non-translating mRNPs. ( A ) Cytoplasmic extracts from uninduced and 12 h K294A-expressing cells were separated on linear sucrose gradients and pooled into mRNP and polysome fractions. ( B ) RNA recovered from the pooled mRNP was spiked with uncapped luciferase RNA as a control for Xrn1 digestion, and capped β-globin RNA as a control for sample recovery. Half of each sample was treated with Xrn1 to degrade uncapped RNAs ( 12 ) and the recovered RNA was assayed by RT-qPCR using primers located near the 5′ ends of luciferase, β-globin, β-actin, RPLP0,VDAC3, ILF2, ZNF207 and MAPK1 mRNA. Results were calculated as in ( 12 ) and are shown as the change in Xrn1 sensitivity of unaffected (β-actin, RPLP0) and target (VDAC3, ILF2, ZNF207 and MAPK1) mRNAs as a function of K294A expression.

    Techniques Used: Expressing, Luciferase, Quantitative RT-PCR

    23) Product Images from "An African tick flavivirus forming an independent clade exhibits unique exoribonuclease-resistant RNA structures in the genomic 3′-untranslated region"

    Article Title: An African tick flavivirus forming an independent clade exhibits unique exoribonuclease-resistant RNA structures in the genomic 3′-untranslated region

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-84365-9

    Xrn1 degradation assay and characterization of the Xrn1-resistant products. ( a , b ) Schematic diagram of predicted secondary structures of two RNA constructs, (+ 31)-xrRNA1 ( a ) and (+ 31)-xrRNA2 ( b ), used for the in vitro Xrn1 degradation assay. The leader sequences in these constructs are artificially designed 31-mers which do not form a prominent structure nor interact with the downstream genomic sequences of xrRNA structures. Heat scale bars represent bits of positional entropy. Black arrows indicate the Xrn1 halt sites. ( c ) In vitro Xrn1 degradation assay using (+31)-xrRNA1 and (+31)-xrRNA2. This image is a part of full-length gel represented in Supplementary Fig. S3a. ( d , e ) Reverse transcription mapping the Xrn1 halt sites with RNA from panel ( c ). The location of the stop site (the 5′ border of the RNA products) is shown with a red arrow to the right, along with the sequence of the RNA surrounding this position. These images are parts of full-length gels represented in Supplementary Fig. S3b,c.
    Figure Legend Snippet: Xrn1 degradation assay and characterization of the Xrn1-resistant products. ( a , b ) Schematic diagram of predicted secondary structures of two RNA constructs, (+ 31)-xrRNA1 ( a ) and (+ 31)-xrRNA2 ( b ), used for the in vitro Xrn1 degradation assay. The leader sequences in these constructs are artificially designed 31-mers which do not form a prominent structure nor interact with the downstream genomic sequences of xrRNA structures. Heat scale bars represent bits of positional entropy. Black arrows indicate the Xrn1 halt sites. ( c ) In vitro Xrn1 degradation assay using (+31)-xrRNA1 and (+31)-xrRNA2. This image is a part of full-length gel represented in Supplementary Fig. S3a. ( d , e ) Reverse transcription mapping the Xrn1 halt sites with RNA from panel ( c ). The location of the stop site (the 5′ border of the RNA products) is shown with a red arrow to the right, along with the sequence of the RNA surrounding this position. These images are parts of full-length gels represented in Supplementary Fig. S3b,c.

    Techniques Used: Degradation Assay, Construct, In Vitro, Genomic Sequencing, Sequencing

    24) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    25) Product Images from "The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA"

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201250

    Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.
    Figure Legend Snippet: Both knocking-down and knocking-out of XRN1 had no effect on the accumulation of sfRNA. XRN1-knockdown (KD) cells were prepared as described in Materials and Methods. Knockdown efficiency was determined by Western blot using anti-XRN1 and anti-β-actin antibodies. The Rel. % value represents the percentage of XRN1 expression in cells transfected with shXRN1 compared to wild-type cells (WT)(100%) as shown at the top (A-C). The XRN1-KD HEK293T (A) or A549 (B) cells were infected with JEV. Total RNA were extracted at the indicated times post infection and Northern blots were done with a DIG-labeled riboprobe detecting nt 10454 to nt 10976 in the 3’UTR (A, D, E) or an IRD 700-labeled JEV(-)10950-10976 probe (B). (C) XRN1-KD HEK293T cells were infected with DENV-2 at an MOI of 5 as a control. Total RNAs were extracted at 72 h post-infection and Northern blots were analyzed using a DIG-labeled riboprobe detecting nt 10270 to nt 10723 in the 3’UTR. Relative amounts of sfRNA were quantified (%) in the XRN1-depleted cells. (D) XRN1-knockout (KO) cells were infected with JEV or DENN-2 at an MOI of 5. RNA isolated from these cells at 48 h post-infection was subjected to Northern blot analysis. (E) RNA degradation analysis of non-replicative 800-nt 3’-terminal monophosphate transcripts derived from genome of JEV or DENV as indicated was measured in vitro by incubating with the indicated amounts of XRN1. Total RNAs extracted from JEV or DENV-2 infected cells (1 μg) were used as the sfRNA size marker (lanes 5 and 10). RNAs were separated by denaturing gel and analyzed by Northern hybridization.

    Techniques Used: Western Blot, Expressing, Transfection, Infection, Northern Blot, Labeling, Knock-Out, Isolation, Derivative Assay, In Vitro, Marker, Hybridization

    Determine the region of 3’ UTR involved in the accumulation of sfRNA constructed in the context of a full-length infectious JEV clone. (A) Predicted secondary structures within the 585-nt 3’ UTR of JEV genome. Nucleotides are numbered from the first base of the genome. The deletion or base substitution regions made in this study are marked or color-coded. (B) Schematic diagram of the 3’ UTR mutants and the results of Northern hybridization for detecting the genome and sfRNA are summarized on the right. (C) BHK-21 cells were infected with recombinant mutant viruses at an MOI of 0.1. RNAs were extracted at 48 h post-infection and Northern hybridization was done as described in Fig 1A . (D) HEK293T cells (WT) or XRN1-knockdown cells (KD) were infected with a PK disrupted mutant (PK1”), and a PK compensatory changed mutant (PK1’1”) viruses at an MOI of 1. Cytoplasmic RNAs were extracted at 60 h post-infection and analyzed by Northern blot. (E) Plaque assays of WT and all mutant viruses were done on BHK-21 cells. Cells were fixed and stained with naphthol blue–black dye at 4 days post-infection. (F) Viral growth kinetics of the WT infectious clone and the recombinant mutant viruses. BHK-21 cells were infected at an MOI of 0.1, and the supernatant fluid of the infected cells was sampled at the indicated times post infection. Titers were determined by plaque assays on BHK-21 cells.
    Figure Legend Snippet: Determine the region of 3’ UTR involved in the accumulation of sfRNA constructed in the context of a full-length infectious JEV clone. (A) Predicted secondary structures within the 585-nt 3’ UTR of JEV genome. Nucleotides are numbered from the first base of the genome. The deletion or base substitution regions made in this study are marked or color-coded. (B) Schematic diagram of the 3’ UTR mutants and the results of Northern hybridization for detecting the genome and sfRNA are summarized on the right. (C) BHK-21 cells were infected with recombinant mutant viruses at an MOI of 0.1. RNAs were extracted at 48 h post-infection and Northern hybridization was done as described in Fig 1A . (D) HEK293T cells (WT) or XRN1-knockdown cells (KD) were infected with a PK disrupted mutant (PK1”), and a PK compensatory changed mutant (PK1’1”) viruses at an MOI of 1. Cytoplasmic RNAs were extracted at 60 h post-infection and analyzed by Northern blot. (E) Plaque assays of WT and all mutant viruses were done on BHK-21 cells. Cells were fixed and stained with naphthol blue–black dye at 4 days post-infection. (F) Viral growth kinetics of the WT infectious clone and the recombinant mutant viruses. BHK-21 cells were infected at an MOI of 0.1, and the supernatant fluid of the infected cells was sampled at the indicated times post infection. Titers were determined by plaque assays on BHK-21 cells.

    Techniques Used: Construct, Northern Blot, Hybridization, Infection, Recombinant, Mutagenesis, Staining

    Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.
    Figure Legend Snippet: Schematic model depicting the mechanism of JEV sfRNA formation. The JEV sfRNA is likely made (i) by transcription initially by RdRp in conjunction with other host factors (HFs), and (ii) the synthesized products could be further trimmed by exoribonuclease XRN1 and/or other unidentified enzymes.

    Techniques Used: Synthesized

    26) Product Images from "Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia"

    Article Title: Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia

    Journal: Viruses

    doi: 10.3390/v12091017

    Xrn1 degradation assay and determination of Xrn1 stop point. ( a ) Schematic diagram of the BJV and BJLV 3′-UTR xrRNA degradation assay. xrRNA structures (shown in the box) were challenged with Xrn1 enzyme. (1) BJV 3′-UTR xrRNA (2) BJLV 3′-UTR xrRNA (3) BJV 3′-UTR xrRNA ΔHP. ( b ) In vitro Xrn1 degradation assay against xrRNA structure of BJV and BJLV, and the hairpin structure of BJLV. Data show a representative result of two independent experiments. Examination of BJV ( c , e ) and BJLV ( d , f ) Xrn1 stop points, which we could unambiguously locate directly at upstream of the xrRNA structures. Red arrow: Stop point.
    Figure Legend Snippet: Xrn1 degradation assay and determination of Xrn1 stop point. ( a ) Schematic diagram of the BJV and BJLV 3′-UTR xrRNA degradation assay. xrRNA structures (shown in the box) were challenged with Xrn1 enzyme. (1) BJV 3′-UTR xrRNA (2) BJLV 3′-UTR xrRNA (3) BJV 3′-UTR xrRNA ΔHP. ( b ) In vitro Xrn1 degradation assay against xrRNA structure of BJV and BJLV, and the hairpin structure of BJLV. Data show a representative result of two independent experiments. Examination of BJV ( c , e ) and BJLV ( d , f ) Xrn1 stop points, which we could unambiguously locate directly at upstream of the xrRNA structures. Red arrow: Stop point.

    Techniques Used: Degradation Assay, In Vitro

    Diagrams of xrRNA structure. xrRNA structure of ( a ) BJV and ( b ) BJLV. Arrows indicate the three-way junction structure and arrowheads indicate Xrn1 stop point; PK: pseudoknot.
    Figure Legend Snippet: Diagrams of xrRNA structure. xrRNA structure of ( a ) BJV and ( b ) BJLV. Arrows indicate the three-way junction structure and arrowheads indicate Xrn1 stop point; PK: pseudoknot.

    Techniques Used:

    27) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ) Representation of the expression vector cassettes (solid lines) used to produce RNA3 and RNA3E species under the control of constitutive G6PDH promoter (black arrowhead). Capped (•) full-length RNAs and ncRNA species are depicted by waved grey and black lines, respectively. Wild-type (wt) ‘core’ sequence is presented in dark and mutated “core” in grey bold lines. The “coremin” motif and its antisense orientation, “nimeroc”, are depicted by black sense and antisense arrows, respectively. Drawings are not to scale and for a better representation of RNA3 species, refer to figure 2 of reference [ 27 ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Northern Blot, Produced, Staining

    A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of
    Figure Legend Snippet: A 55 nt core fragment of RNA3 of BNYVV represses Xrn1. ( A ) A radiolabeled RNA containing a 5′ monophosphate (Reporter) was incubated with purified recombinant XRN1 for the times indicated. A 20X molar excess of lightly radiolabeled, 5′ monophosphorylated non-specific competitor RNA (‘Control RNA’ lanes), a competitor transcript containing the 55 nt core BNYVV RNA3 fragment (‘BNYVV-55mer’ lanes), or a competitor transcript containing the 3′ UTR of Dengue virus type 2 (‘DENV 3′ UTR’ lanes) was added to reactions. After the times indicated, reaction products were analyzed on 5% polyacrylamide gels containing urea and visualized by phosphorimaging. ( B ) Graphical presentation of the effect of the various competitor RNAs on Xrn1 activity on the Reporter transcript. Results shown are from three independent experiments. The asterisk represents a p value of

    Techniques Used: Incubation, Purification, Recombinant, Activity Assay

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.
    Figure Legend Snippet: Accumulation of noncoding RNA3sf from chimeric RNA3sf inhibits Xrn1 and AtXRN4 exoribonucleases in Saccharomyces cerevisiae . ( A ) The ‘core’ sequence was replaced by a wt (sf) or mutated (pk1) flavivirus sequence to produce RNA3sf and RNA3pk1, respectively. PK1 pseudoknot involving stem-loop II (SL-II) is shown; ( B ) northern blot analyses of RNA3 and ncRNA species produced in yeasts. Plasmids allowing the expression of RNA species indicated that RNA3, RNA3E, RNA3sf, RNA3pk1, or empty vector (Ø) were introduced in wt yeasts (lanes 1–5) or Xrn1-defective yeasts (Δ xrn1 , lanes 6–9) complemented with a vector allowing for the production of yeast Xrn1 (lanes 6, 8, and 9) or plant XRN4 (lane 7). The RNA3* and ncRNA3* are similar to RNA3 and ncRNA3, respectively, but possess a CYC1 terminator sequence followed by a polyA tail (see Figure 1 and text for details). Positions of the RNA species are indicated on the right. Total RNAs were subjected to northern blot analysis using a BNYVV-specific 3′ probe complementary to nt 1277–1774. The partial complementarity of the probe with the ncRNA3sf species does not allow for quantitative comparisons. Loading was visualized by ethidium-bromide staining (rRNA). No sample was loaded between lanes 8 and 9.

    Techniques Used: Sequencing, Northern Blot, Produced, Expressing, Plasmid Preparation, Staining

    28) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    29) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    30) Product Images from "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells"

    Article Title: The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

    Journal: bioRxiv

    doi: 10.1101/852434

    Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .
    Figure Legend Snippet: Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .

    Techniques Used: Recombinant, In Vitro, Produced, Purification, Polyacrylamide Gel Electrophoresis

    31) Product Images from "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells"

    Article Title: The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

    Journal: bioRxiv

    doi: 10.1101/852434

    Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .
    Figure Legend Snippet: Susceptibility of short transcripts to recombinant hDcp2 in vitro. Short 25-nt transcripts were produced by IVT, followed by 3’ end trimming by DNAzyme 10-23 and removal of uncapped RNAs by 5’-polyphosphatase and Xrn1 treatment. Purified RNA (30 ng each) was subjected to treatment with hDcp2 (10 nM) for 60 min. Aliquots taken at indicated time points were resolved by PAGE and bands corresponding to capped and uncapped RNAs were quantified densitometrically. (A) Representative PAGE analyses obtained for m 7 GpppApG-RNA and m 7 GpppA m pG-RNA. Arrows indicate the bands used to quantify the amount of the substrate (green) and the product (blue). The raw data from triplicate experiment for all studied RNAs is shown in Figure S8; (B-F) Quantitative results for all studied RNAs. The fraction of capped RNA remaining in the total RNA was plotted as a function of time. Data points represent mean values ± SD from triplicate experiments. Lines between the data points were added as a guide to the eye. The numerical values for time point 15 min are shown in Table 3 .

    Techniques Used: Recombinant, In Vitro, Produced, Purification, Polyacrylamide Gel Electrophoresis

    32) Product Images from "Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity"

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Journal: Viruses

    doi: 10.3390/v10030137

    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Figure Legend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Techniques Used: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.
    Figure Legend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Techniques Used: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).
    Figure Legend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Techniques Used: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.
    Figure Legend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    Related Articles

    other:

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity
    Article Snippet: Kinetics was followed for 12 h. Parallel experiments were carried out with a mixture of wt RNA3 (3 µg) and RNA3E (3 µg) in the presence of 4 U of RppH and 8 U of Xrn1.

    Article Title: The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA
    Article Snippet: Although stalling of XRN1 by the JEV sfRNA has been reported previously [ ], detailed mechanism of the sfRNA formation has not yet been elucidated.

    Article Title: RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening
    Article Snippet: The RNAs were then digested with XRN-1, a 5′ to 3′ exoribonuclease that acts on RNAs only with a 5′ monophosphate group.

    Purification:

    Article Title: Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms
    Article Snippet: In case of Scd6 and Edc3, purified proteins/domains were dialyzed in 20 mM Tris, 100 mM NaCl and 10% glycerol. .. Purified yeast Xrn1 was purchased from NEB. .. Purification of the Lsm1-7 complex was performed using Flag purification (Sigma) from yeast cells grown to mid-log phase in minimal media (6-8L) using the pat1Δ lsm1Δ strain with Lsm5-His6 (yRP2758) harboring a plasmid expressing Flag-Lsm1 (pRP1911).

    Derivative Assay:

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity
    Article Snippet: We next complemented our mapping approach using in vitro transcripts with similar 5′ extremities starting at position 1222 of BNYVV RNA3 and possessing 16 (B-1), 44 (B-2), or 55 (B-3) nucleotides downstream in an Xrn1 decay assay performed using a short time course with an amount of highly active recombinant Kluyveromyces lactis Xrn1 enzyme similar to the amount of RNA substrate in the reaction as previously described [ , ]. .. As seen in A,B, while a similar sized control RNA derived from pGEM-4 vector sequences failed to stall Xrn1 (at the site indicated by the arrow in B), the 55 nt long ‘B-3’ transcript was able to effectively stall Xrn1. .. Shortening the BNYVV-derived sequences from the 3′ end by 10 (B-2) or more (B-3) bases completely abrogated the ability of the transcript to stall Xrn1 and generate a decay intermediate ( B).

    Plasmid Preparation:

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity
    Article Snippet: We next complemented our mapping approach using in vitro transcripts with similar 5′ extremities starting at position 1222 of BNYVV RNA3 and possessing 16 (B-1), 44 (B-2), or 55 (B-3) nucleotides downstream in an Xrn1 decay assay performed using a short time course with an amount of highly active recombinant Kluyveromyces lactis Xrn1 enzyme similar to the amount of RNA substrate in the reaction as previously described [ , ]. .. As seen in A,B, while a similar sized control RNA derived from pGEM-4 vector sequences failed to stall Xrn1 (at the site indicated by the arrow in B), the 55 nt long ‘B-3’ transcript was able to effectively stall Xrn1. .. Shortening the BNYVV-derived sequences from the 3′ end by 10 (B-2) or more (B-3) bases completely abrogated the ability of the transcript to stall Xrn1 and generate a decay intermediate ( B).

    Incubation:

    Article Title: Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection
    Article Snippet: After extraction, the RNA samples were subjected to enzymatic degradation via use of the 5′ to 3′ exonuclease XRN-1, which is capable of degrading 5′ monophosphate and RNAs in vitro but is unable to effectively degrade RNA substrates that are 5′ capped ( , ). .. Briefly, equivalent amounts of viral genomic RNAs were incubated in the presence of 0.25 units of XRN-1 (NEB) in a final volume of 20 μl for a period of 5 min at 37°C prior to being quenched via the addition of high-salt column buffer (25 mM Tris-HCl [pH 7.6]–400 mM NaCl–0.1% [wt/vol] sodium dodecyl sulfate [SDS]). .. After the termination of the reaction, the RNAs were purified via phenol-chloroform extraction and ethanol precipitation with linear acrylamide carrier.

    Produced:

    Article Title: Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia
    Article Snippet: 3.7. xrRNA Structure of BJV and BJLV Can Block Xrn1 EnzymeOur in silico analysis suggested that both BJV and BJLV have a three-way junction element that is structurally homologous to xrRNAs known in MBFVs. .. To investigate whether the xrRNA structures of BJV and BJLV have the same capability to stall Xrn1 we challenged the xrRNA structure (BJV 3′-UTR xrRNA and BJLV 3′-UTR xrRNA ∆HP), which were produced by in vitro transcription, with Xrn1 enzyme [ a(1,3)]. ..

    In Vitro:

    Article Title: Discoveries of Exoribonuclease-Resistant Structures of Insect-Specific Flaviviruses Isolated in Zambia
    Article Snippet: 3.7. xrRNA Structure of BJV and BJLV Can Block Xrn1 EnzymeOur in silico analysis suggested that both BJV and BJLV have a three-way junction element that is structurally homologous to xrRNAs known in MBFVs. .. To investigate whether the xrRNA structures of BJV and BJLV have the same capability to stall Xrn1 we challenged the xrRNA structure (BJV 3′-UTR xrRNA and BJLV 3′-UTR xrRNA ∆HP), which were produced by in vitro transcription, with Xrn1 enzyme [ a(1,3)]. ..

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    New England Biolabs xrn1
    A 55 nt fragment containing the coremin motif is sufficient to stall <t>XRN1.</t> ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
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    A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Journal: Viruses

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    doi: 10.3390/v10030137

    Figure Lengend Snippet: A 55 nt fragment containing the coremin motif is sufficient to stall XRN1. ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.

    Article Snippet: As seen in A,B, while a similar sized control RNA derived from pGEM-4 vector sequences failed to stall Xrn1 (at the site indicated by the arrow in B), the 55 nt long ‘B-3’ transcript was able to effectively stall Xrn1.

    Techniques: Derivative Assay, Sequencing, Incubation, Purification, Recombinant, Acrylamide Gel Assay

    Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Journal: Viruses

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    doi: 10.3390/v10030137

    Figure Lengend Snippet: Both ncRNA3sf and ncRNA3pk1 stall Xrn1 processing in vitro. ( A ) Schematic representation of the T7-driven cDNA clones constructs used to produce run-off in vitro transcripts depicted in waved lines. Double and single arrowheads correspond to the sf and pk1 structural motifs, respectively. The position of the restriction sites used and the size of the transcripts are indicated; ( B ) 5′ phosphorylated chimeric RNA3sf or RNA3pk1 and ( C ) BN3sf or BN3pk1 species were mixed with commercial Xrn1 enzyme for 6 h. Aliquots were sampled at the time indicated and RNAs species were detected by northern blot using a specific DNA probe able to reveal both full-length and ncRNA3 species.

    Article Snippet: As seen in A,B, while a similar sized control RNA derived from pGEM-4 vector sequences failed to stall Xrn1 (at the site indicated by the arrow in B), the 55 nt long ‘B-3’ transcript was able to effectively stall Xrn1.

    Techniques: In Vitro, Clone Assay, Construct, Northern Blot

    Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Journal: Viruses

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    doi: 10.3390/v10030137

    Figure Lengend Snippet: Exoribonucleases are responsible for noncoding RNA3 species accumulation in Saccharomyces cerevisiae . ( A ]. RNA sizes are presented on the right; ( B , C ) Northern blot analyses of RNA3 and ncRNA species produced in yeasts; ( B ) vectors producing wt RNA3, mutated RNA3E, or control vector (Ø) were introduced in S. cerevisiae (lanes 1–3) or Xrn1 defective strain (Δ xrn1 ) (lanes 4–5). Expression of RNA3 (lanes 6–10) was performed in ∆ xrn1 strain together with an empty vector (EV, lane 6) or vectors expressing: yeast Xrn1 (lane 7), plant ATXRN4 (XRN4, lanes 8 and 9), or a defective Xrn1 enzyme (Xrn1cat, lane 10). Total RNAs were subjected to northern blot analysis using a beet necrotic yellow vein virus (BNYVV)-specific 3′ probe complementary to nt 1277–1774; ( C ) total RNAs from yeast expressing RNA3 were differentially visualized using two probes (lanes 1 and 2). RNA3, RNA3*, ncRNA3, and ncRNA3* were revealed with a coremin-specific probe (lane 1) while RNA3* and ncRNA3* appeared only when the CYC1 terminator-specific probe was used (lane 2). Black triangles indicate the two full-length RNA3 species. Loadings were visualized by ethidium-bromide staining (rRNA).

    Article Snippet: As seen in A,B, while a similar sized control RNA derived from pGEM-4 vector sequences failed to stall Xrn1 (at the site indicated by the arrow in B), the 55 nt long ‘B-3’ transcript was able to effectively stall Xrn1.

    Techniques: Northern Blot, Produced, Plasmid Preparation, Expressing, Staining

    Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Journal: Viruses

    Article Title: Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    doi: 10.3390/v10030137

    Figure Lengend Snippet: Characterization of the minimal RNA3 3′ domain required for the efficient stalling of the Xrn1 enzyme. ( A ) Representation of the fragments obtained using T7 promoter containing primer (T7-RNA3F) and reverse primers (positions specified by blue boxes) cloned into pUC57 that served as templates for the production of 1R to 9R transcripts, ranging from 989 to 558 nt, possessing the same 5′ extremity with decreasing 3′ end length. The arrowhead locates the 5′ position of the ncRNA3 species (nt 1234). The expected product size after RppH and Xrn1 treatments are specified. Construct 6R was not obtained; ( B ) after 6 h of RppH/Xrn1 treatment, RNAs were analyzed by a 24-cm long run on 1.5% denaturing-agarose gel followed by northern blot using a radiolabeled DNA oligomer probe complementary to the “coremin” sequence. The ncRNA3 transcripts and 1R transcripts were treated with RppH alone or with RppH and Xrn1. The lengths of some RNA species are specified on the left side.

    Article Snippet: As seen in A,B, while a similar sized control RNA derived from pGEM-4 vector sequences failed to stall Xrn1 (at the site indicated by the arrow in B), the 55 nt long ‘B-3’ transcript was able to effectively stall Xrn1.

    Techniques: Clone Assay, Construct, Agarose Gel Electrophoresis, Northern Blot, Sequencing

    ) Dhh1, B) Scd6, C) Xrn1, D) Lsm1-7 complex, E) Dcp1, F) Dcp2, and G) Pat1 C domain with the GST moiety cleaved with PreScission protease. The

    Journal: Molecular cell

    Article Title: Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms

    doi: 10.1016/j.molcel.2010.08.025

    Figure Lengend Snippet: ) Dhh1, B) Scd6, C) Xrn1, D) Lsm1-7 complex, E) Dcp1, F) Dcp2, and G) Pat1 C domain with the GST moiety cleaved with PreScission protease. The

    Article Snippet: Purified yeast Xrn1 was purchased from NEB.

    Techniques:

    YTHDF1 interacts with ZAP, DDX17, and DCP2 Venn diagram of YTHDF1 interaction partners in CNE2EBV, HeLa, and HEK293 cells. The interactions between YTHDF1 and ZAP (B) or DDX17 (C) were detected by co‐IP assays in 293T cells. The results were detected using the indicated antibodies. YTHDF1 did not interact with XRN1 according to the co‐IP assay in 293T cells. The interaction of YTHDF1 with DCP2 detected by co‐IP assays in 293T cells. Co‐localization of the endogenous YTHDF1 with ZAP, DDX17, and DCP2 in HK1 cells detected by immunofluorescence. YTHDF1 was stained with the mouse monoclonal YTHDF1 antibody and an Alexa Fluor 594‐conjugated secondary antibody. ZAP, DDX17, and DCP2 were stained with the target‐specific rabbit primary antibodies and an Alexa Fluor 488‐conjugated secondary antibody. The nuclei were counterstained with DAPI (blue). The merged signal of YTHDF1 and ZAP, DDX17, or DCP2 is shown in yellow. Representative of three independent experiments are shown. Scale bars: 5 μm. The right panel shows the results of co‐localization analysis in the selected region analyzed under a confocal laser scanning microscope. r : correlation coefficients. The P ‐values were calculated using Pearson’s correlation test. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: N(6)‐methyladenosine‐binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay

    doi: 10.15252/embr.202050128

    Figure Lengend Snippet: YTHDF1 interacts with ZAP, DDX17, and DCP2 Venn diagram of YTHDF1 interaction partners in CNE2EBV, HeLa, and HEK293 cells. The interactions between YTHDF1 and ZAP (B) or DDX17 (C) were detected by co‐IP assays in 293T cells. The results were detected using the indicated antibodies. YTHDF1 did not interact with XRN1 according to the co‐IP assay in 293T cells. The interaction of YTHDF1 with DCP2 detected by co‐IP assays in 293T cells. Co‐localization of the endogenous YTHDF1 with ZAP, DDX17, and DCP2 in HK1 cells detected by immunofluorescence. YTHDF1 was stained with the mouse monoclonal YTHDF1 antibody and an Alexa Fluor 594‐conjugated secondary antibody. ZAP, DDX17, and DCP2 were stained with the target‐specific rabbit primary antibodies and an Alexa Fluor 488‐conjugated secondary antibody. The nuclei were counterstained with DAPI (blue). The merged signal of YTHDF1 and ZAP, DDX17, or DCP2 is shown in yellow. Representative of three independent experiments are shown. Scale bars: 5 μm. The right panel shows the results of co‐localization analysis in the selected region analyzed under a confocal laser scanning microscope. r : correlation coefficients. The P ‐values were calculated using Pearson’s correlation test. Source data are available online for this figure.

    Article Snippet: Briefly, the cytoplasmic RNA samples (3 µg) were treated with three units of 5′‐phosphate‐dependent exonuclease XRN1 (New England BioLabs, USA) for 2 h at 37°C according to the manufacturer’s protocol.

    Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Laser-Scanning Microscopy

    Features of lncRNA1840 and 1459. Analysis of expression of lncRNA1840 (A) and lncRNA1459 (B) during fruit ripening. Actin expression values were used for internal reference. The relative level of lncRNA transcripts was normalized to that at the IM stage of AC fruits where the amount was arbitrarily assigned a value of 1. Error bars indicate ±SD of three biological replicates, each measured in triplicate. IM, immature green; MG, mature green; BR, breaker; PK, pink; RR, red-ripe stage. (C) Determination of the 3′ end structure of lncRNAs. Random-primed RT–PCR was performed on total RNAs, poly(A) + , and poly(A) – RNAs from tomato fruits to detect novel lncRNAs. (D) Analysis of the 5′ end structure of lncRNAs. Total RNAs from tomato fruits were treated (+) or not (–) with various enzymes and subjected to random-primed RT–PCR to detect specific lncRNAs. RppH, 5′ pyrophosphohydrolase; XRN-1, 5′ to 3′ exoribonuclease; PNK, polynucleotide kinase. –RT, reverse transcription was performed in the absence of reverse transcriptase. Transcript from RIN was detected by RT–PCR as a control for poly(A) + RNA and capped RNA. PCRs with genomic DNA were used as positive controls. The amplification region of RIN primers contained one intron which results in a larger PCR product of DNA template than the others.

    Journal: Journal of Experimental Botany

    Article Title: RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening

    doi: 10.1093/jxb/erv203

    Figure Lengend Snippet: Features of lncRNA1840 and 1459. Analysis of expression of lncRNA1840 (A) and lncRNA1459 (B) during fruit ripening. Actin expression values were used for internal reference. The relative level of lncRNA transcripts was normalized to that at the IM stage of AC fruits where the amount was arbitrarily assigned a value of 1. Error bars indicate ±SD of three biological replicates, each measured in triplicate. IM, immature green; MG, mature green; BR, breaker; PK, pink; RR, red-ripe stage. (C) Determination of the 3′ end structure of lncRNAs. Random-primed RT–PCR was performed on total RNAs, poly(A) + , and poly(A) – RNAs from tomato fruits to detect novel lncRNAs. (D) Analysis of the 5′ end structure of lncRNAs. Total RNAs from tomato fruits were treated (+) or not (–) with various enzymes and subjected to random-primed RT–PCR to detect specific lncRNAs. RppH, 5′ pyrophosphohydrolase; XRN-1, 5′ to 3′ exoribonuclease; PNK, polynucleotide kinase. –RT, reverse transcription was performed in the absence of reverse transcriptase. Transcript from RIN was detected by RT–PCR as a control for poly(A) + RNA and capped RNA. PCRs with genomic DNA were used as positive controls. The amplification region of RIN primers contained one intron which results in a larger PCR product of DNA template than the others.

    Article Snippet: The RNAs were then digested with XRN-1, a 5′ to 3′ exoribonuclease that acts on RNAs only with a 5′ monophosphate group.

    Techniques: Expressing, Random Primed, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction