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  • 98
    New England Biolabs xrn1
    A 55 nt fragment containing the coremin motif is sufficient to stall <t>XRN1.</t> ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    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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    95
    New England Biolabs xrn 1
    A 55 nt fragment containing the coremin motif is sufficient to stall <t>XRN1.</t> ( A ) 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or a 101-nt RNA containing a 55 base fragment from the RNA3 segment of BNYVV (nts 1222–1277) at the 3′ end and 53 nts of pGEM-4 polylinker sequence at its 5′ end to serve as a landing site for 5′-to-3′ exonucleases) were incubated with either purified recombinant XRN1 from Kluyveromyces lactis (rXrn1 panel) or cytoplasmic extract from C6/36 Aedes albopictus cells for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. ( B ) Top: sequence of the 55 nts BNYVV RNA fragment. The black arrow indicates the site of XRN1 stalling. Fragments B-1, B-2, and B-3 containing BNYVV-specific sequences ranging from position 1222 to the base indicated in the figure. Bottom: 5′ monophosphorylated radiolabeled RNA substrates containing either control sequences derived from pGEM-4 or the B-1, B-2, or B-3 fragments of the RNA3 segment (nts 1222–1277) as indicated in the top part of the panel (inserted into the pGEM-4 polylinker as indicated in panel A) were incubated with purified recombinant XRN1 for the times indicated. Reaction products were resolved on a 5% acrylamide gel containing urea and viewed by phosphorimaging. Arrows indicate the positions of the RNA species stalling XRN1.
    Xrn 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    xrn1  (Bethyl)
    88
    Bethyl xrn1
    <t>XRN1</t> regulates ex-miRNA decay in recipient cells ( A ) Relative quantification analysis of ex-miRNA-223-3p in A549 cells. siXRN1-transfected A549 cells co-cultured with PMN overnight were harvested at the indicated periods of time post PMN removal (Time Washed, T.W.). Results are representative of three biological replicates. In the right panel, immunoblot analysis of XRN1 expression. b-ACTIN served as an equal loading control. ( B ) Immunoblot analysis of FOXO1 and EMT marker expression levels. β-ACTIN served as an equal loading control. ( C ) In vitro invasion assay of siXRN1-transfected A549 cells. A549 cells co-cultured with SPN of PMN, produced in serum-free medium, were seeded in the upper part of transwells. The number of cells attached to the bottom of a Matrigel-coated membrane after 16 h was quantified after crystal violet staining. Data represent the quantification of five biological replicates, ‘centre values’ as mean and error bars as s.d. * for P
    Xrn1, supplied by Bethyl, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bethyl rabbit anti xrn1 antibody affinity purified
    Deletions of C-terminal UPF1 phosphorylation sites impair NMD differently. ( A ) UPF1 protein architecture is depicted schematically. All structural and functional domains are indicated; the presence of potential phosphorylation sites (SQ/TQ) are shown in red and blue, respectively. ( B , C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected LacZ-H4 ( B ) or β-globin ( C ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the <t>XRN1</t> control knockdown. Representative Western blots are shown at the bottom , using tubulin as loading control.
    Rabbit Anti Xrn1 Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Viruses

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

    doi: 10.3390/v10030137

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

    Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

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

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

    Journal: Viruses

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

    doi: 10.3390/v10030137

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

    Article Snippet: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

    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: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

    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: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

    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: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

    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: As seen in A, while the presence of a 20X excess of a non-specific control RNA in the reaction had no effect on Xrn1 activity on a radiolabeled reporter RNA, the accumulation of ncRNA species due to Xrn1 stalling on either the BNYVV 55 nt RNA or the DENV 3′ UTR transcript correlated with an inhibition of the substrate degradation.

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

    XRN1 regulates ex-miRNA decay in recipient cells ( A ) Relative quantification analysis of ex-miRNA-223-3p in A549 cells. siXRN1-transfected A549 cells co-cultured with PMN overnight were harvested at the indicated periods of time post PMN removal (Time Washed, T.W.). Results are representative of three biological replicates. In the right panel, immunoblot analysis of XRN1 expression. b-ACTIN served as an equal loading control. ( B ) Immunoblot analysis of FOXO1 and EMT marker expression levels. β-ACTIN served as an equal loading control. ( C ) In vitro invasion assay of siXRN1-transfected A549 cells. A549 cells co-cultured with SPN of PMN, produced in serum-free medium, were seeded in the upper part of transwells. The number of cells attached to the bottom of a Matrigel-coated membrane after 16 h was quantified after crystal violet staining. Data represent the quantification of five biological replicates, ‘centre values’ as mean and error bars as s.d. * for P

    Journal: Nucleic Acids Research

    Article Title: Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition

    doi: 10.1093/nar/gkw1284

    Figure Lengend Snippet: XRN1 regulates ex-miRNA decay in recipient cells ( A ) Relative quantification analysis of ex-miRNA-223-3p in A549 cells. siXRN1-transfected A549 cells co-cultured with PMN overnight were harvested at the indicated periods of time post PMN removal (Time Washed, T.W.). Results are representative of three biological replicates. In the right panel, immunoblot analysis of XRN1 expression. b-ACTIN served as an equal loading control. ( B ) Immunoblot analysis of FOXO1 and EMT marker expression levels. β-ACTIN served as an equal loading control. ( C ) In vitro invasion assay of siXRN1-transfected A549 cells. A549 cells co-cultured with SPN of PMN, produced in serum-free medium, were seeded in the upper part of transwells. The number of cells attached to the bottom of a Matrigel-coated membrane after 16 h was quantified after crystal violet staining. Data represent the quantification of five biological replicates, ‘centre values’ as mean and error bars as s.d. * for P

    Article Snippet: Mechanistically, we demonstrated that the ex-miR-223-3p are extremely labile miRNA, rapidly degraded by XRN1 following removal of EVs producing PMN.

    Techniques: Transfection, Cell Culture, Expressing, Marker, In Vitro, Invasion Assay, Produced, Staining

    Deletions of C-terminal UPF1 phosphorylation sites impair NMD differently. ( A ) UPF1 protein architecture is depicted schematically. All structural and functional domains are indicated; the presence of potential phosphorylation sites (SQ/TQ) are shown in red and blue, respectively. ( B , C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected LacZ-H4 ( B ) or β-globin ( C ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. Representative Western blots are shown at the bottom , using tubulin as loading control.

    Journal: RNA

    Article Title: Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates

    doi: 10.1261/rna.059659.116

    Figure Lengend Snippet: Deletions of C-terminal UPF1 phosphorylation sites impair NMD differently. ( A ) UPF1 protein architecture is depicted schematically. All structural and functional domains are indicated; the presence of potential phosphorylation sites (SQ/TQ) are shown in red and blue, respectively. ( B , C ) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected LacZ-H4 ( B ) or β-globin ( C ) served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. Representative Western blots are shown at the bottom , using tubulin as loading control.

    Article Snippet: The antibodies against tubulin (T6074) and Flag (F7425) were from Sigma-Aldrich, the antibody against SMG6 (ab87539) was from Abcam, the antibodies against XRN1 (A300-443A) and SMG7 (A302-170A) were from Bethyl, and the antibody against UPF1 was kindly provided by Jens Lykke-Andersen.

    Techniques: Functional Assay, Northern Blot, Transfection, Construct, Western Blot

    Functional analysis of potential endogenous NMD targets identified during high-throughput sequencing. ( A – D ) A PCR-based approach was used to quantify 3′ fragment levels upon knockdown of NMD effectors. HeLa cells were transiently transfected with the indicated siRNAs and poly(A) + RNA was extracted. 3′ Decay intermediates were ligated to an RNA linker, followed by reverse transcription with an oligo(dT) primer and gene-specific PCR. Overall transcript levels were determined with primer pairs located downstream from the estimated endocleavage site (second panel each, indicated as “gene int.”). PCR with TATA-Box binding protein (TBP) primers was used for cDNA level determination. For each class of NMD targets ( A , B , C , D, respectively), the same set of cDNA was used and therefore the same TBP profiles are shown for each target type. Degradome-sequencing reads were plotted against their position on the indicated mRNAs. Endocleavage events within the selected targets were visualized in an enlarged view spanning 150 nt (±75 nt). The position of the second nucleotide of the respective stop codon is set as zero. Mapped reads per nucleotide were plotted against the mRNA length for each knockdown condition (Luciferase [Luc, black], XRN1 [orange], XRN1/SMG6 [blue]). ( A ) PDRG1, SURF6, and MED10 transcripts with a long 3′ UTR. ( B ) IFRD1 and BAG1 transcripts with an uORF. ( C ) C11orf31, encoding for Selenoprotein H, containing a selenocysteine (Sec) codon. ( D ) TMEM222, incorporation of an alternative exon (indicated in purple), harboring a PTC as well as RPL10A, a transcript gaining a PTC probably due to alternative splice site usage within the third intron.

    Journal: RNA

    Article Title: Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates

    doi: 10.1261/rna.059659.116

    Figure Lengend Snippet: Functional analysis of potential endogenous NMD targets identified during high-throughput sequencing. ( A – D ) A PCR-based approach was used to quantify 3′ fragment levels upon knockdown of NMD effectors. HeLa cells were transiently transfected with the indicated siRNAs and poly(A) + RNA was extracted. 3′ Decay intermediates were ligated to an RNA linker, followed by reverse transcription with an oligo(dT) primer and gene-specific PCR. Overall transcript levels were determined with primer pairs located downstream from the estimated endocleavage site (second panel each, indicated as “gene int.”). PCR with TATA-Box binding protein (TBP) primers was used for cDNA level determination. For each class of NMD targets ( A , B , C , D, respectively), the same set of cDNA was used and therefore the same TBP profiles are shown for each target type. Degradome-sequencing reads were plotted against their position on the indicated mRNAs. Endocleavage events within the selected targets were visualized in an enlarged view spanning 150 nt (±75 nt). The position of the second nucleotide of the respective stop codon is set as zero. Mapped reads per nucleotide were plotted against the mRNA length for each knockdown condition (Luciferase [Luc, black], XRN1 [orange], XRN1/SMG6 [blue]). ( A ) PDRG1, SURF6, and MED10 transcripts with a long 3′ UTR. ( B ) IFRD1 and BAG1 transcripts with an uORF. ( C ) C11orf31, encoding for Selenoprotein H, containing a selenocysteine (Sec) codon. ( D ) TMEM222, incorporation of an alternative exon (indicated in purple), harboring a PTC as well as RPL10A, a transcript gaining a PTC probably due to alternative splice site usage within the third intron.

    Article Snippet: The antibodies against tubulin (T6074) and Flag (F7425) were from Sigma-Aldrich, the antibody against SMG6 (ab87539) was from Abcam, the antibodies against XRN1 (A300-443A) and SMG7 (A302-170A) were from Bethyl, and the antibody against UPF1 was kindly provided by Jens Lykke-Andersen.

    Techniques: Functional Assay, Next-Generation Sequencing, Polymerase Chain Reaction, Transfection, Binding Assay, Sequencing, Luciferase, Size-exclusion Chromatography

    SMG7 abundance influences endocleavage efficiency for long 3′ UTR targets. ( A ) Domain structure of SMG7, showing the N-terminal 14-3-3-like domain (interacts with UPF1) and α-helical extensions as well as the C-terminal PC region (interacts with POP2). ( B ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected β-globin served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control. ( C ) Schematic representation of the transfected triosephosphate isomerase (TPI) reporter with a PTC at amino acid position 160. Exons are depicted as white (untranslated) or black (translated) boxes, introns as two connecting black lines, and the Northern probe binding sites as light gray boxes. Vector derived 5′ UTR intron and SV40 poly(A) signal (pA) are shown. ( D ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs using LacZ-4H as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control.

    Journal: RNA

    Article Title: Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates

    doi: 10.1261/rna.059659.116

    Figure Lengend Snippet: SMG7 abundance influences endocleavage efficiency for long 3′ UTR targets. ( A ) Domain structure of SMG7, showing the N-terminal 14-3-3-like domain (interacts with UPF1) and α-helical extensions as well as the C-terminal PC region (interacts with POP2). ( B ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Cotransfected β-globin served as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control. ( C ) Schematic representation of the transfected triosephosphate isomerase (TPI) reporter with a PTC at amino acid position 160. Exons are depicted as white (untranslated) or black (translated) boxes, introns as two connecting black lines, and the Northern probe binding sites as light gray boxes. Vector derived 5′ UTR intron and SV40 poly(A) signal (pA) are shown. ( D ) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs, plasmids, and reporter constructs using LacZ-4H as control mRNA. Endocleavage products (3′ fragments) are indicated. Mean values of reporter and 3′ fragment signal ± SD ( n = 3) were quantified and normalized to the XRN1 control knockdown. A representative Western blot is shown at the bottom , using tubulin as loading control.

    Article Snippet: The antibodies against tubulin (T6074) and Flag (F7425) were from Sigma-Aldrich, the antibody against SMG6 (ab87539) was from Abcam, the antibodies against XRN1 (A300-443A) and SMG7 (A302-170A) were from Bethyl, and the antibody against UPF1 was kindly provided by Jens Lykke-Andersen.

    Techniques: Northern Blot, Transfection, Construct, Western Blot, Binding Assay, Plasmid Preparation, Derivative Assay