xrn1  (New England Biolabs)


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    Structured Review

    New England Biolabs xrn1
    Both knocking-down and knocking-out of <t>XRN1</t> 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.
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    Images

    1) 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

    2) 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

    3) 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

    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 "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

    6) 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

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    New England Biolabs xrn1
    Both knocking-down and knocking-out of <t>XRN1</t> 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.
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs yeast xrn1
    ) Dhh1, B) Scd6, C) <t>Xrn1,</t> D) Lsm1-7 complex, E) Dcp1, F) Dcp2, and G) Pat1 C domain with the GST moiety cleaved with PreScission protease. The
    Yeast Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: PLoS ONE

    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

    doi: 10.1371/journal.pone.0201250

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: PLoS ONE

    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

    doi: 10.1371/journal.pone.0201250

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: PLoS ONE

    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

    doi: 10.1371/journal.pone.0201250

    Figure Lengend 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.

    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.

    Techniques: Synthesized

    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: 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.

    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: 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.

    Techniques: 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).

    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 ) 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).

    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.

    Techniques: 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

    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 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

    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.

    Techniques: 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.

    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: 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.

    Techniques: 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.

    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: 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.

    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.

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

    ) 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:

    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.

    Journal: Viruses

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

    doi: 10.3390/v12091017

    Figure Lengend 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.

    Article Snippet: 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)].

    Techniques: 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.

    Journal: Viruses

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

    doi: 10.3390/v12091017

    Figure Lengend 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.

    Article Snippet: 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)].

    Techniques: