yeast xrn1  (New England Biolabs)


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    Structured Review

    New England Biolabs yeast xrn1
    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and <t>Xrn1</t> depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.
    Yeast Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    yeast xrn1 - by Bioz Stars, 2023-02
    86/100 stars

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    1) Product Images from "mRNA decay can be uncoupled from deadenylation during stress response"

    Article Title: mRNA decay can be uncoupled from deadenylation during stress response

    Journal: bioRxiv

    doi: 10.1101/2023.01.20.524924

    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.
    Figure Legend Snippet: Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.

    Techniques Used: In Vivo, Binding Assay, Expressing, In Vitro

    A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.
    Figure Legend Snippet: A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.

    Techniques Used: Western Blot, Produced, In Vitro

    xrn1  (New England Biolabs)


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    Structured Review

    New England Biolabs xrn1
    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and <t>Xrn1</t> depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xrn1/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xrn1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "mRNA decay can be uncoupled from deadenylation during stress response"

    Article Title: mRNA decay can be uncoupled from deadenylation during stress response

    Journal: bioRxiv

    doi: 10.1101/2023.01.20.524924

    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.
    Figure Legend Snippet: Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.

    Techniques Used: In Vivo, Binding Assay, Expressing, In Vitro

    A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.
    Figure Legend Snippet: A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.

    Techniques Used: Western Blot, Produced, In Vitro

    xrn1  (New England Biolabs)


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    Structured Review

    New England Biolabs xrn1
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xrn1/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
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    xrn1 - by Bioz Stars, 2023-02
    86/100 stars

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    xrn1 efficiency  (New England Biolabs)


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    Structured Review

    New England Biolabs xrn1 efficiency
    Xrn1 Efficiency, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xrn1 efficiency/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xrn1 efficiency - by Bioz Stars, 2023-02
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    recombinant xrn1  (New England Biolabs)


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    New England Biolabs recombinant xrn1
    ( A ) Digestion of 2′-phosphorylated RNA by <t>Xrn1</t> yields a product that inhibits further degradation. ( B ) The 2′-phosphate on tRNA-Pro-UGG can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . In the lower panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. Asterisks correspond to previously described but uncharacterized Pro-TGG splicing intermediates , . ( C ) Unmodified, 2′-phosphorylated, and 2′-O-methylated RNA substrates show zero, substantial, and trace resistance to Xrn1 digestion, respectively. ( D ) A 2′-phosphate inhibits decay by the 5′→3′ exonucleases Xrn1, Dxo1, and RNase J1. The location of the 2′-phosphorylated linkage is indicated by the red “G” in the substrate at right, with inferred stalling sites for each exonuclease marked with arrows. ( E ) In budding yeast, the unfolded protein response (UPR) initiates a noncanonical splicing event on the mRNA HAC1 during which the endonuclease Ire1 excises the HAC1 intron (thin line), followed by exon ligation by Trl1 (black and gray boxes), generating an internal 2′-phosphate at the splice junction that is subsequently removed by the 2′-phosphotransferase Tpt1. ( F ) RNA from WT and xrn1Δ tpt1Δ (10x-tRNA) cells was treated with DMSO or tunicamycin and left untreated (left panel) or enzymatically and digested (right panel) followed by nanopore sequencing. 5′-end alignments of reads are expressed in counts per million reads (CPM). ( G ) Subtraction of undigested background signal from signal from the Tm-treated, <t>rXrn1-digested</t> libraries in ( F ), yields a predominant 5′-end signal on HAC1 s at the splice junction (yellow box). At right, an enhanced view of this region shows the major peak in wild-type cells is located precisely at the exon-exon junction (position 727), whereas the end signal in the mutant is 13 nucleotides downstream, an offset consistent with premature termination of base calling when a bona fide 5′ RNA end exits a nanopore. ( H ) The wild-type rXrn1 enrichment scores above have been subtracted from the mutant scores at each nucleotide position.
    Recombinant Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant xrn1/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant xrn1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "Direct detection of RNA repair by nanopore sequencing"

    Article Title: Direct detection of RNA repair by nanopore sequencing

    Journal: bioRxiv

    doi: 10.1101/2022.05.29.493267

    ( A ) Digestion of 2′-phosphorylated RNA by Xrn1 yields a product that inhibits further degradation. ( B ) The 2′-phosphate on tRNA-Pro-UGG can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . In the lower panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. Asterisks correspond to previously described but uncharacterized Pro-TGG splicing intermediates , . ( C ) Unmodified, 2′-phosphorylated, and 2′-O-methylated RNA substrates show zero, substantial, and trace resistance to Xrn1 digestion, respectively. ( D ) A 2′-phosphate inhibits decay by the 5′→3′ exonucleases Xrn1, Dxo1, and RNase J1. The location of the 2′-phosphorylated linkage is indicated by the red “G” in the substrate at right, with inferred stalling sites for each exonuclease marked with arrows. ( E ) In budding yeast, the unfolded protein response (UPR) initiates a noncanonical splicing event on the mRNA HAC1 during which the endonuclease Ire1 excises the HAC1 intron (thin line), followed by exon ligation by Trl1 (black and gray boxes), generating an internal 2′-phosphate at the splice junction that is subsequently removed by the 2′-phosphotransferase Tpt1. ( F ) RNA from WT and xrn1Δ tpt1Δ (10x-tRNA) cells was treated with DMSO or tunicamycin and left untreated (left panel) or enzymatically and digested (right panel) followed by nanopore sequencing. 5′-end alignments of reads are expressed in counts per million reads (CPM). ( G ) Subtraction of undigested background signal from signal from the Tm-treated, rXrn1-digested libraries in ( F ), yields a predominant 5′-end signal on HAC1 s at the splice junction (yellow box). At right, an enhanced view of this region shows the major peak in wild-type cells is located precisely at the exon-exon junction (position 727), whereas the end signal in the mutant is 13 nucleotides downstream, an offset consistent with premature termination of base calling when a bona fide 5′ RNA end exits a nanopore. ( H ) The wild-type rXrn1 enrichment scores above have been subtracted from the mutant scores at each nucleotide position.
    Figure Legend Snippet: ( A ) Digestion of 2′-phosphorylated RNA by Xrn1 yields a product that inhibits further degradation. ( B ) The 2′-phosphate on tRNA-Pro-UGG can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . In the lower panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. Asterisks correspond to previously described but uncharacterized Pro-TGG splicing intermediates , . ( C ) Unmodified, 2′-phosphorylated, and 2′-O-methylated RNA substrates show zero, substantial, and trace resistance to Xrn1 digestion, respectively. ( D ) A 2′-phosphate inhibits decay by the 5′→3′ exonucleases Xrn1, Dxo1, and RNase J1. The location of the 2′-phosphorylated linkage is indicated by the red “G” in the substrate at right, with inferred stalling sites for each exonuclease marked with arrows. ( E ) In budding yeast, the unfolded protein response (UPR) initiates a noncanonical splicing event on the mRNA HAC1 during which the endonuclease Ire1 excises the HAC1 intron (thin line), followed by exon ligation by Trl1 (black and gray boxes), generating an internal 2′-phosphate at the splice junction that is subsequently removed by the 2′-phosphotransferase Tpt1. ( F ) RNA from WT and xrn1Δ tpt1Δ (10x-tRNA) cells was treated with DMSO or tunicamycin and left untreated (left panel) or enzymatically and digested (right panel) followed by nanopore sequencing. 5′-end alignments of reads are expressed in counts per million reads (CPM). ( G ) Subtraction of undigested background signal from signal from the Tm-treated, rXrn1-digested libraries in ( F ), yields a predominant 5′-end signal on HAC1 s at the splice junction (yellow box). At right, an enhanced view of this region shows the major peak in wild-type cells is located precisely at the exon-exon junction (position 727), whereas the end signal in the mutant is 13 nucleotides downstream, an offset consistent with premature termination of base calling when a bona fide 5′ RNA end exits a nanopore. ( H ) The wild-type rXrn1 enrichment scores above have been subtracted from the mutant scores at each nucleotide position.

    Techniques Used: Northern Blot, Recombinant, In Vitro, Methylation, Ligation, Nanopore Sequencing, Mutagenesis

    ( A ) Unmodified 20 nt oligo and 2′-phosphorylated 26 nt oligo used in this experiment. ( B ) 26 nt oligos were 3’-end labeled with radiolabeled pCp prior to incubation with or without rXrn1. After digestion, all samples were treated with phenol:chloroform and ethanol precipitated, and then mixed with equimolar amounts of the 20 nt oligo, followed by a 1 hour incubation with or without T4 RNL1. Ligation products are visible in lane 2, but undetectable in lane 4.
    Figure Legend Snippet: ( A ) Unmodified 20 nt oligo and 2′-phosphorylated 26 nt oligo used in this experiment. ( B ) 26 nt oligos were 3’-end labeled with radiolabeled pCp prior to incubation with or without rXrn1. After digestion, all samples were treated with phenol:chloroform and ethanol precipitated, and then mixed with equimolar amounts of the 20 nt oligo, followed by a 1 hour incubation with or without T4 RNL1. Ligation products are visible in lane 2, but undetectable in lane 4.

    Techniques Used: Labeling, Incubation, Ligation

    ( A ) Cloverleaf diagram of a spliced tRNA from tpt1Δ cells, indicating the 2′-phosphorylated nucleotide located immediately 3′ of the anticodon, and the 5mer centered on this position in gray. ( B ) The RNA modification landscape within the indicated 5mer in ( A ) for each intron-containing tRNA in S. cerevisiae . Blue boxes indicate positions with known tRNA modifications in the MODOMICS database. ( C ) The 2′-phosphate on tRNA-Trp-CCA can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . 3′-exon degradation intermediates accumulate in tpt1Δ (10x-tRNA) cells, while two larger exonuclease-resistant bands are also present, albeit less abundant, in RNA from trl1Δ (10x-tRNA) cells treated with rXrn1. Given that the MODOMICS database lists annotated methyl groups on this mature tRNA at nucleotides 31 and 33 , and that 2′-O-methyl groups also inhibit Xrn1 , albeit substantially less robustly than 2′-phosphates , it is possible that these larger bands are produced by 2′-O-methyl-mediated rXrn1 inhibition. However, in the context of a trl1Δ tpt1Δ double mutant, these degradation intermediates disappear, raising the possibility of additional crosstalk between tRNA splicing and tRNA modification pathways. In the lower sub-panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. ( D ) IGV snapshots of direct tRNA sequencing reads mapping to intron-containing S. cerevisiae tRNAs. Each colored bar represents a position with >20% mismatching to the reference base; gray bars indicate positions which did not exceed this threshold. Numbers in brackets at right indicate the Y axis range in read counts, and tRNA anticodons are surrounded by a black box. Modified positions reported in MODOMICS are annotated below the reference sequence. To the right of select IGV alignment views are northern blots of total RNA from S. cerevisiae strains treated with and without recombinant Xrn1 in vitro , using a 3′-exon hybridizing probe.
    Figure Legend Snippet: ( A ) Cloverleaf diagram of a spliced tRNA from tpt1Δ cells, indicating the 2′-phosphorylated nucleotide located immediately 3′ of the anticodon, and the 5mer centered on this position in gray. ( B ) The RNA modification landscape within the indicated 5mer in ( A ) for each intron-containing tRNA in S. cerevisiae . Blue boxes indicate positions with known tRNA modifications in the MODOMICS database. ( C ) The 2′-phosphate on tRNA-Trp-CCA can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . 3′-exon degradation intermediates accumulate in tpt1Δ (10x-tRNA) cells, while two larger exonuclease-resistant bands are also present, albeit less abundant, in RNA from trl1Δ (10x-tRNA) cells treated with rXrn1. Given that the MODOMICS database lists annotated methyl groups on this mature tRNA at nucleotides 31 and 33 , and that 2′-O-methyl groups also inhibit Xrn1 , albeit substantially less robustly than 2′-phosphates , it is possible that these larger bands are produced by 2′-O-methyl-mediated rXrn1 inhibition. However, in the context of a trl1Δ tpt1Δ double mutant, these degradation intermediates disappear, raising the possibility of additional crosstalk between tRNA splicing and tRNA modification pathways. In the lower sub-panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. ( D ) IGV snapshots of direct tRNA sequencing reads mapping to intron-containing S. cerevisiae tRNAs. Each colored bar represents a position with >20% mismatching to the reference base; gray bars indicate positions which did not exceed this threshold. Numbers in brackets at right indicate the Y axis range in read counts, and tRNA anticodons are surrounded by a black box. Modified positions reported in MODOMICS are annotated below the reference sequence. To the right of select IGV alignment views are northern blots of total RNA from S. cerevisiae strains treated with and without recombinant Xrn1 in vitro , using a 3′-exon hybridizing probe.

    Techniques Used: Modification, Northern Blot, Recombinant, In Vitro, Produced, Inhibition, Mutagenesis, Sequencing

    xrn1  (New England Biolabs)


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    New England Biolabs xrn1
    A Northern blot detection of sfRNAs produced by ISFs. For PaRV, PCV, BinJV and HVV C6/36 cells were infected at MOI = 1. Total RNA was isolated at 5 dpi. For KRBV, total RNA was isolated from virus-positive and virus-negative (Mock) Anopheles mosquitoes. RNA was then used for Northern blotting with the probe complementary to the last 25nt of viral 3’UTRs. B The effect of <t>XRN1</t> knock-down on the production of sfRNAs by ISFs. Aag2 cells were transfected with dsRNA against Aedes aegypti XRN1 (dsXRN1) or GFP (dsNC) and infected with respective viruses at MOI = 1 at 24hpt. At 48hpi, total RNA was isolated from the cells and used for Northern blotting as in ( A ). Bottom panels represent the Et-Br staining of the gels used for Northern transfer with 7SL cellular RNA visualised as a loading control. C In vitro XRN1 resistance assay with ISF 3’UTRs. RNA corresponding to 3’UTRs of ISFs was transcribed in vitro, briefly heated and then refolded by gradual cooling to 28 °C or placed on ice to preserve the denatured state. Samples were then treated with purified XRN1 and RppH (to convert 5’PPP into 5’P) and analysed by electrophoresis in denaturing PAAG. Gels were stained with ethidium bromide (Et-Br). All images are representative of at least two independent experiments that produced similar results.
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    1) Product Images from "Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinants of sfRNA biogenesis and provides new insights into flavivirus evolution"

    Article Title: Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinants of sfRNA biogenesis and provides new insights into flavivirus evolution

    Journal: Nature Communications

    doi: 10.1038/s41467-022-28977-3

    A Northern blot detection of sfRNAs produced by ISFs. For PaRV, PCV, BinJV and HVV C6/36 cells were infected at MOI = 1. Total RNA was isolated at 5 dpi. For KRBV, total RNA was isolated from virus-positive and virus-negative (Mock) Anopheles mosquitoes. RNA was then used for Northern blotting with the probe complementary to the last 25nt of viral 3’UTRs. B The effect of XRN1 knock-down on the production of sfRNAs by ISFs. Aag2 cells were transfected with dsRNA against Aedes aegypti XRN1 (dsXRN1) or GFP (dsNC) and infected with respective viruses at MOI = 1 at 24hpt. At 48hpi, total RNA was isolated from the cells and used for Northern blotting as in ( A ). Bottom panels represent the Et-Br staining of the gels used for Northern transfer with 7SL cellular RNA visualised as a loading control. C In vitro XRN1 resistance assay with ISF 3’UTRs. RNA corresponding to 3’UTRs of ISFs was transcribed in vitro, briefly heated and then refolded by gradual cooling to 28 °C or placed on ice to preserve the denatured state. Samples were then treated with purified XRN1 and RppH (to convert 5’PPP into 5’P) and analysed by electrophoresis in denaturing PAAG. Gels were stained with ethidium bromide (Et-Br). All images are representative of at least two independent experiments that produced similar results.
    Figure Legend Snippet: A Northern blot detection of sfRNAs produced by ISFs. For PaRV, PCV, BinJV and HVV C6/36 cells were infected at MOI = 1. Total RNA was isolated at 5 dpi. For KRBV, total RNA was isolated from virus-positive and virus-negative (Mock) Anopheles mosquitoes. RNA was then used for Northern blotting with the probe complementary to the last 25nt of viral 3’UTRs. B The effect of XRN1 knock-down on the production of sfRNAs by ISFs. Aag2 cells were transfected with dsRNA against Aedes aegypti XRN1 (dsXRN1) or GFP (dsNC) and infected with respective viruses at MOI = 1 at 24hpt. At 48hpi, total RNA was isolated from the cells and used for Northern blotting as in ( A ). Bottom panels represent the Et-Br staining of the gels used for Northern transfer with 7SL cellular RNA visualised as a loading control. C In vitro XRN1 resistance assay with ISF 3’UTRs. RNA corresponding to 3’UTRs of ISFs was transcribed in vitro, briefly heated and then refolded by gradual cooling to 28 °C or placed on ice to preserve the denatured state. Samples were then treated with purified XRN1 and RppH (to convert 5’PPP into 5’P) and analysed by electrophoresis in denaturing PAAG. Gels were stained with ethidium bromide (Et-Br). All images are representative of at least two independent experiments that produced similar results.

    Techniques Used: Northern Blot, Produced, Infection, Isolation, Transfection, Staining, In Vitro, Purification, Electrophoresis

    A Secondary structure of BinJV 3’UTR generated by SHAPE. SL – stem-loop, DB – dumbbell, RCS3 – reverse conserved sequence 3, CS3 – conserved sequence 3, PK – pseudoknot. B Secondary structures of BinJV stem-loops. C Structure-based sequence alignment between BinJV and MBF xrRNAs. D XRN1 resistance assay with WT and mutated BinJV 3’UTRs. Mutations PK1’ (GAGAG- > CUCUC), PK2’ (UGGUUG- > ACCAAC), nPK1’ (UAGCG- > AUCGC) and nPK2’ (GCGUC- > CGCAG) were introduced into the terminal loop regions of the corresponding stem-loops. The image is representative of four independent experiments that produced similar results. E Densitometry analysis of ( D ). The values are the means of four independent experiments ± SD. Statistical analysis is two-sided one-way ANOVA. F Consensus structure of dISF xrRNAs built based on the covariance model. Covarying base pairs are highlighted in green ( G ) Secondary structure of HVV xrRNA generated by SHAPE.
    Figure Legend Snippet: A Secondary structure of BinJV 3’UTR generated by SHAPE. SL – stem-loop, DB – dumbbell, RCS3 – reverse conserved sequence 3, CS3 – conserved sequence 3, PK – pseudoknot. B Secondary structures of BinJV stem-loops. C Structure-based sequence alignment between BinJV and MBF xrRNAs. D XRN1 resistance assay with WT and mutated BinJV 3’UTRs. Mutations PK1’ (GAGAG- > CUCUC), PK2’ (UGGUUG- > ACCAAC), nPK1’ (UAGCG- > AUCGC) and nPK2’ (GCGUC- > CGCAG) were introduced into the terminal loop regions of the corresponding stem-loops. The image is representative of four independent experiments that produced similar results. E Densitometry analysis of ( D ). The values are the means of four independent experiments ± SD. Statistical analysis is two-sided one-way ANOVA. F Consensus structure of dISF xrRNAs built based on the covariance model. Covarying base pairs are highlighted in green ( G ) Secondary structure of HVV xrRNA generated by SHAPE.

    Techniques Used: Generated, Sequencing, Produced

    A Secondary structure of PaRV 3’UTR generated using SHAPE-assisted folding. Colour intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of PaRV xrRNAs. Each structural element is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PaRV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the CAC - > GUG change in the PK-forming regions. Image is representative from three independent experiments that showed similar results. D Structure-based alignment of PaRV xrRNAs was performed using LocARNA.
    Figure Legend Snippet: A Secondary structure of PaRV 3’UTR generated using SHAPE-assisted folding. Colour intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of PaRV xrRNAs. Each structural element is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PaRV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the CAC - > GUG change in the PK-forming regions. Image is representative from three independent experiments that showed similar results. D Structure-based alignment of PaRV xrRNAs was performed using LocARNA.

    Techniques Used: Generated, In Vitro

    A Secondary structure of PCV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK – pseudoknot. B Secondary structure of PCV xrRNAs. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PCV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the GAC - > CUG change in the PK-forming region. Image is representative from three independent experiments that showed similar results. D Alignment of sequence and structure of PCV xrRNAs was performed using LocARNA.
    Figure Legend Snippet: A Secondary structure of PCV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK – pseudoknot. B Secondary structure of PCV xrRNAs. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated PCV 3’UTRs. PK1’, PK2’ and PK3’ mutations were introduced into the terminal loop regions of SLI, SLII and SLIII, respectively, and represented the GAC - > CUG change in the PK-forming region. Image is representative from three independent experiments that showed similar results. D Alignment of sequence and structure of PCV xrRNAs was performed using LocARNA.

    Techniques Used: Generated, In Vitro, Sequencing

    A Secondary structure of KRBV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of KRBV xrRNA. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated KRBV 3’UTRs. PK1’ mutation was GUUGC - > CAACG change of PK-forming nucleotides in the L2 loop of the SLI. The experiment was repeated three times with similar results. D Sequence alignment of KRBV and AnFV1 xrRNAs. The colour coding matches ( B ) and shows conserved structural elements. E Predicted secondary structure of AnFV1 xrRNA. The conserved structural elements are shown in colours that match ( D ).
    Figure Legend Snippet: A Secondary structure of KRBV 3’UTR generated using SHAPE-assisted folding. Shading intensity indicates NMIA reactivity. Values are the means from 3 independent experiments, each with technical triplicates. SL – stem loop, PK -pseudoknot. B Secondary structure of KRBV xrRNA. Each element of the secondary structure is shown in colour. C In vitro XRN1 resistance assay with WT and mutated KRBV 3’UTRs. PK1’ mutation was GUUGC - > CAACG change of PK-forming nucleotides in the L2 loop of the SLI. The experiment was repeated three times with similar results. D Sequence alignment of KRBV and AnFV1 xrRNAs. The colour coding matches ( B ) and shows conserved structural elements. E Predicted secondary structure of AnFV1 xrRNA. The conserved structural elements are shown in colours that match ( D ).

    Techniques Used: Generated, In Vitro, Mutagenesis, Sequencing

    xrn1  (New England Biolabs)


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    New England Biolabs xrn1
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    xrn1  (New England Biolabs)


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    New England Biolabs xrn1
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    recombinant xrn1  (New England Biolabs)


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    a Schematic representation of processes involved in 5′-to-3′ RNA degradation. Eukaryotic and bacterial triphosphorylated messenger (m)RNA are processed by proteins DCP2 and RppH, respectively, to generate monophosphorylated substrates for the 5′-3′ exonuclease <t>XRN1,</t> RNase E or RNase J. We hypothesize that a mammalian Nudix hydrolase could prepare viral RNA for degradation by removing pyrophosphate groups. b Hep3B cells with a CRISPR/Cas9-mediated KO of XRN1 or Hep3B cells transduced with a non-targeting-control vector (NTC) were infected with VSV-GFP at MOI 0.01 for 48 h. GFP expression was continuously quantified by live-cell microscopy. The data points are displayed as the mean of five technical replicates ± SD. One representative experiment of three is shown. c HeLa cells transfected with siRNA pools targeting the indicated Nudix gene for 48 h were infected with VSV-GFP ( x -axis) or VSV-AV3-GFP ( y -axis). Twenty-four hours after infection, GFP expression was analyzed by fluorometric analysis. The displayed z -scores were calculated from three independent reactions. d HeLa cells were treated with siRNA pools for 48 h targeting the transcript of NUDT2 , NUDT12 , NUDT14 , NUDT17 , or non-targeting siRNA control (siScrambled) and infected with Influenza A reporter virus expressing the renilla luciferase (IAV-ren) at MOI 1. Forty-eight hours post-infection, renilla luciferase activity was analyzed. The means of three biological replicates ± SD are shown; ** p = 0.004, * p = 0.03 as analyzed by two-way analysis of variance (ANOVA) statistics. RLU relative light units. n.s. not significant.
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    1) Product Images from "NUDT2 initiates viral RNA degradation by removal of 5′-phosphates"

    Article Title: NUDT2 initiates viral RNA degradation by removal of 5′-phosphates

    Journal: Nature Communications

    doi: 10.1038/s41467-021-27239-y

    a Schematic representation of processes involved in 5′-to-3′ RNA degradation. Eukaryotic and bacterial triphosphorylated messenger (m)RNA are processed by proteins DCP2 and RppH, respectively, to generate monophosphorylated substrates for the 5′-3′ exonuclease XRN1, RNase E or RNase J. We hypothesize that a mammalian Nudix hydrolase could prepare viral RNA for degradation by removing pyrophosphate groups. b Hep3B cells with a CRISPR/Cas9-mediated KO of XRN1 or Hep3B cells transduced with a non-targeting-control vector (NTC) were infected with VSV-GFP at MOI 0.01 for 48 h. GFP expression was continuously quantified by live-cell microscopy. The data points are displayed as the mean of five technical replicates ± SD. One representative experiment of three is shown. c HeLa cells transfected with siRNA pools targeting the indicated Nudix gene for 48 h were infected with VSV-GFP ( x -axis) or VSV-AV3-GFP ( y -axis). Twenty-four hours after infection, GFP expression was analyzed by fluorometric analysis. The displayed z -scores were calculated from three independent reactions. d HeLa cells were treated with siRNA pools for 48 h targeting the transcript of NUDT2 , NUDT12 , NUDT14 , NUDT17 , or non-targeting siRNA control (siScrambled) and infected with Influenza A reporter virus expressing the renilla luciferase (IAV-ren) at MOI 1. Forty-eight hours post-infection, renilla luciferase activity was analyzed. The means of three biological replicates ± SD are shown; ** p = 0.004, * p = 0.03 as analyzed by two-way analysis of variance (ANOVA) statistics. RLU relative light units. n.s. not significant.
    Figure Legend Snippet: a Schematic representation of processes involved in 5′-to-3′ RNA degradation. Eukaryotic and bacterial triphosphorylated messenger (m)RNA are processed by proteins DCP2 and RppH, respectively, to generate monophosphorylated substrates for the 5′-3′ exonuclease XRN1, RNase E or RNase J. We hypothesize that a mammalian Nudix hydrolase could prepare viral RNA for degradation by removing pyrophosphate groups. b Hep3B cells with a CRISPR/Cas9-mediated KO of XRN1 or Hep3B cells transduced with a non-targeting-control vector (NTC) were infected with VSV-GFP at MOI 0.01 for 48 h. GFP expression was continuously quantified by live-cell microscopy. The data points are displayed as the mean of five technical replicates ± SD. One representative experiment of three is shown. c HeLa cells transfected with siRNA pools targeting the indicated Nudix gene for 48 h were infected with VSV-GFP ( x -axis) or VSV-AV3-GFP ( y -axis). Twenty-four hours after infection, GFP expression was analyzed by fluorometric analysis. The displayed z -scores were calculated from three independent reactions. d HeLa cells were treated with siRNA pools for 48 h targeting the transcript of NUDT2 , NUDT12 , NUDT14 , NUDT17 , or non-targeting siRNA control (siScrambled) and infected with Influenza A reporter virus expressing the renilla luciferase (IAV-ren) at MOI 1. Forty-eight hours post-infection, renilla luciferase activity was analyzed. The means of three biological replicates ± SD are shown; ** p = 0.004, * p = 0.03 as analyzed by two-way analysis of variance (ANOVA) statistics. RLU relative light units. n.s. not significant.

    Techniques Used: CRISPR, Transduction, Plasmid Preparation, Infection, Expressing, Microscopy, Transfection, Luciferase, Activity Assay

    a In vitro transcribed PPP-renilla RNA was left untreated (mock) or incubated with 600 nM NUDT2, 1 U XRN1, 600 nM NUDT2 together with 1 U XRN1, 5 U RppH, or 5 U RppH together with 1 U XRN1 and 10 U RNase for 4 h. The remaining RNA was quantified by RT-qPCR. The bar plot shows the fold change compared to input RNA as the mean ± SD of three technical replicates. b In vitro synthesized HCV RNA was treated with 5 U RNase, 600 nM NUDT2, 1 U XRN1, or co-treated with 600 nM NUDT2 and 1 U XRN1 and then analyzed on an agarose gel. c In vitro transcribed HCV RNA encoding luciferase was electroporated into HeLa CRISPR KO cells lacking NUDT2 or HeLa cells treated with a non-targeting-control vector (NTC). Viral RNA load was quantified 4 and 6 h post electroporation by the abundance of firefly luciferase RNA. d In vitro transcribed triphosphorylated renilla RNA and capped firefly luciferase RNA were co-electroporated into Hep3B CRISPR/Cas9 KO cells lacking NUDT2 , XRN1 , or both NUDT2 and XRN1 , or Hep3B cells treated with a non-targeting-control vector (NTC). RNA load was quantified 1 and 4 h post electroporation by quantifying the abundance of firefly luciferase or renilla RNA. e Hep3B treated with CRISPR/Cas9 lentivectors targeting NUDT2 , XRN1 , NUDT2 , and XRN1 or with a non-targeting-control vector (NTC) were infected with VSV-GFP at MOI 0.01. GFP expression was monitored using automated live-cell microscopy. The data points are displayed as the mean of five technical replicates ± SD. Shown is one representative experiment of three independent experiments.
    Figure Legend Snippet: a In vitro transcribed PPP-renilla RNA was left untreated (mock) or incubated with 600 nM NUDT2, 1 U XRN1, 600 nM NUDT2 together with 1 U XRN1, 5 U RppH, or 5 U RppH together with 1 U XRN1 and 10 U RNase for 4 h. The remaining RNA was quantified by RT-qPCR. The bar plot shows the fold change compared to input RNA as the mean ± SD of three technical replicates. b In vitro synthesized HCV RNA was treated with 5 U RNase, 600 nM NUDT2, 1 U XRN1, or co-treated with 600 nM NUDT2 and 1 U XRN1 and then analyzed on an agarose gel. c In vitro transcribed HCV RNA encoding luciferase was electroporated into HeLa CRISPR KO cells lacking NUDT2 or HeLa cells treated with a non-targeting-control vector (NTC). Viral RNA load was quantified 4 and 6 h post electroporation by the abundance of firefly luciferase RNA. d In vitro transcribed triphosphorylated renilla RNA and capped firefly luciferase RNA were co-electroporated into Hep3B CRISPR/Cas9 KO cells lacking NUDT2 , XRN1 , or both NUDT2 and XRN1 , or Hep3B cells treated with a non-targeting-control vector (NTC). RNA load was quantified 1 and 4 h post electroporation by quantifying the abundance of firefly luciferase or renilla RNA. e Hep3B treated with CRISPR/Cas9 lentivectors targeting NUDT2 , XRN1 , NUDT2 , and XRN1 or with a non-targeting-control vector (NTC) were infected with VSV-GFP at MOI 0.01. GFP expression was monitored using automated live-cell microscopy. The data points are displayed as the mean of five technical replicates ± SD. Shown is one representative experiment of three independent experiments.

    Techniques Used: In Vitro, Incubation, Quantitative RT-PCR, Synthesized, Agarose Gel Electrophoresis, Luciferase, CRISPR, Plasmid Preparation, Electroporation, Infection, Expressing, Microscopy

    xrn1  (New England Biolabs)


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    Structured Review

    New England Biolabs xrn1
    EV-associated and cellular GAPDH mRNA differ in size and polyadenylation status. ( a ) Total RNA from ES-2 cells and their corresponding EVs were isolated, followed by RT-PCR. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used. Markers (200, 100 bp). ( b ) RNA was isolated from ES-2 cells and their corresponding EVs. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used, and GAPDH mRNA was quantified by RT-qPCR. ( c ) Detection of GAPDH mRNA in EVs by the glyoxal Northern blot analysis. Total cellular and EV-RNA (300 ng) were analyzed by glyoxal agarose gel electrophoresis and Northern blotting. Exposure time 3 min ( d ) Total RNA from ES-2-derived EVs was isolated and treated with <t>Xrn1</t> exoribonuclease (+Xrn1). As control, no enzyme was added (−Xrn1). RT-PCR was performed using primers directed against transcripts indicated. Markers (200, 100 bp).
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    Images

    1) Product Images from "Molecular Determinants for RNA Release into Extracellular Vesicles"

    Article Title: Molecular Determinants for RNA Release into Extracellular Vesicles

    Journal: Cells

    doi: 10.3390/cells10102674

    EV-associated and cellular GAPDH mRNA differ in size and polyadenylation status. ( a ) Total RNA from ES-2 cells and their corresponding EVs were isolated, followed by RT-PCR. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used. Markers (200, 100 bp). ( b ) RNA was isolated from ES-2 cells and their corresponding EVs. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used, and GAPDH mRNA was quantified by RT-qPCR. ( c ) Detection of GAPDH mRNA in EVs by the glyoxal Northern blot analysis. Total cellular and EV-RNA (300 ng) were analyzed by glyoxal agarose gel electrophoresis and Northern blotting. Exposure time 3 min ( d ) Total RNA from ES-2-derived EVs was isolated and treated with Xrn1 exoribonuclease (+Xrn1). As control, no enzyme was added (−Xrn1). RT-PCR was performed using primers directed against transcripts indicated. Markers (200, 100 bp).
    Figure Legend Snippet: EV-associated and cellular GAPDH mRNA differ in size and polyadenylation status. ( a ) Total RNA from ES-2 cells and their corresponding EVs were isolated, followed by RT-PCR. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used. Markers (200, 100 bp). ( b ) RNA was isolated from ES-2 cells and their corresponding EVs. For reverse transcription, either oligo(dT) or random hexamers (dT, dN 6 ) were used, and GAPDH mRNA was quantified by RT-qPCR. ( c ) Detection of GAPDH mRNA in EVs by the glyoxal Northern blot analysis. Total cellular and EV-RNA (300 ng) were analyzed by glyoxal agarose gel electrophoresis and Northern blotting. Exposure time 3 min ( d ) Total RNA from ES-2-derived EVs was isolated and treated with Xrn1 exoribonuclease (+Xrn1). As control, no enzyme was added (−Xrn1). RT-PCR was performed using primers directed against transcripts indicated. Markers (200, 100 bp).

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Northern Blot, Agarose Gel Electrophoresis, Derivative Assay

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    New England Biolabs yeast xrn1
    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and <t>Xrn1</t> depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.
    Yeast Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs xrn1
    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and <t>Xrn1</t> depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.
    Xrn1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs xrn1 efficiency
    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and <t>Xrn1</t> depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.
    Xrn1 Efficiency, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs recombinant xrn1
    ( A ) Digestion of 2′-phosphorylated RNA by <t>Xrn1</t> yields a product that inhibits further degradation. ( B ) The 2′-phosphate on tRNA-Pro-UGG can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . In the lower panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. Asterisks correspond to previously described but uncharacterized Pro-TGG splicing intermediates , . ( C ) Unmodified, 2′-phosphorylated, and 2′-O-methylated RNA substrates show zero, substantial, and trace resistance to Xrn1 digestion, respectively. ( D ) A 2′-phosphate inhibits decay by the 5′→3′ exonucleases Xrn1, Dxo1, and RNase J1. The location of the 2′-phosphorylated linkage is indicated by the red “G” in the substrate at right, with inferred stalling sites for each exonuclease marked with arrows. ( E ) In budding yeast, the unfolded protein response (UPR) initiates a noncanonical splicing event on the mRNA HAC1 during which the endonuclease Ire1 excises the HAC1 intron (thin line), followed by exon ligation by Trl1 (black and gray boxes), generating an internal 2′-phosphate at the splice junction that is subsequently removed by the 2′-phosphotransferase Tpt1. ( F ) RNA from WT and xrn1Δ tpt1Δ (10x-tRNA) cells was treated with DMSO or tunicamycin and left untreated (left panel) or enzymatically and digested (right panel) followed by nanopore sequencing. 5′-end alignments of reads are expressed in counts per million reads (CPM). ( G ) Subtraction of undigested background signal from signal from the Tm-treated, <t>rXrn1-digested</t> libraries in ( F ), yields a predominant 5′-end signal on HAC1 s at the splice junction (yellow box). At right, an enhanced view of this region shows the major peak in wild-type cells is located precisely at the exon-exon junction (position 727), whereas the end signal in the mutant is 13 nucleotides downstream, an offset consistent with premature termination of base calling when a bona fide 5′ RNA end exits a nanopore. ( H ) The wild-type rXrn1 enrichment scores above have been subtracted from the mutant scores at each nucleotide position.
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    Image Search Results


    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.

    Journal: bioRxiv

    Article Title: mRNA decay can be uncoupled from deadenylation during stress response

    doi: 10.1101/2023.01.20.524924

    Figure Lengend Snippet: Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.

    Article Snippet: Purified Tt Xrn1 was inspected in 10% SDS-PAGE stained with Coomassie Brilliant Blue R-250, along with commercial yeast Xrn1 (NEB; M0338) as a positive control (Supplementary Figure X).

    Techniques: In Vivo, Binding Assay, Expressing, In Vitro

    A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.

    Journal: bioRxiv

    Article Title: mRNA decay can be uncoupled from deadenylation during stress response

    doi: 10.1101/2023.01.20.524924

    Figure Lengend Snippet: A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.

    Article Snippet: Purified Tt Xrn1 was inspected in 10% SDS-PAGE stained with Coomassie Brilliant Blue R-250, along with commercial yeast Xrn1 (NEB; M0338) as a positive control (Supplementary Figure X).

    Techniques: Western Blot, Produced, In Vitro

    Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.

    Journal: bioRxiv

    Article Title: mRNA decay can be uncoupled from deadenylation during stress response

    doi: 10.1101/2023.01.20.524924

    Figure Lengend Snippet: Pab1 is required for in vivo deadenylation and decapping commonly occurs at steady-state mRNA pA-tail lengths. A . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells. On the right side of the graph, schematic representations of the Pab1 polyA-binding protein are shown to outline that the region bound by one molecule is around 22-30 As. B . Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the exponential model relative to the transcript deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 2 hours of Pab1 depletion using the AID system. D . Graph shows the global pA-tail length distribution of coding transcripts in control cells compared to Dcp2 and Xrn1 depletion using AID. The distribution shows all pA-tail lengths regardless of transcript abundance, thus mRNAs of high expression are overrepresented. E . Scheme shows that Xrn1 can only digest mRNAs devoid of a cap, potentially altering pA-tail distribution if transcripts are only decapped at a defined pA-tail length. F . Graph shows the global pA-tail length distribution of coding transcripts in control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ . G-H . Graphs show pA-tail length distributions of HCH1 mRNA in (G) control cells compared to wild-type samples digested in vitro with Xrn1 and in pop2Δ and for (H) control cells compared to Dcp2 and Xrn1 depletion using AID. I-J . Same as (G-H) for YMR122W-A mRNA.

    Article Snippet: Purified Tt Xrn1 was inspected in 10% SDS-PAGE stained with Coomassie Brilliant Blue R-250, along with commercial yeast Xrn1 (NEB; M0338) as a positive control (Supplementary Figure X).

    Techniques: In Vivo, Binding Assay, Expressing, In Vitro

    A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.

    Journal: bioRxiv

    Article Title: mRNA decay can be uncoupled from deadenylation during stress response

    doi: 10.1101/2023.01.20.524924

    Figure Lengend Snippet: A. Scatterplot shows the quantile-specific deadenylation rate estimate calculated using the multiple fit linear model relative to the transcripts deadenylation rate mean obtained by averaging deadenylation rates of the upper quantiles (75 th – 95 th ). B . Western blot shows depletion of Pab1 protein using the auxin-inducible degron for 1 and 2 hours. Western blot analysis for Rpb3 protein was used as a loading control. C . Scatterplot shows the change in mRNA abundance on the x-axis relative to the absolute change in the mean pA-tail length of mRNAs upon 1 hour Pab1 depletion using the AID system. D . Scatterplot shows the distribution of pA-tail quantile values in wild-type cells compared to Pab1-depletion using AID system for 1 and 2 hours. The thin grey dots show individual mRNA pA-tail lengths in control cells, while the larger circles show the median values in both the control (black dot) and Pab1-depleted cells (empty red and blue circles). E . Western blot shows the efficiency of depletion of Dcp2 using the AID system for 2 hours. The PGK1 blot is shown as a loading control. F . Growth test produced using serial 10-fold dilutions of cells showing depletion of Dcp2 and Xrn1 using the AID system gives the expected growth phenotype. G . Bottom panel shows an autoradiogram of the bulk mRNA pA-tail length distributions in control cells and strains depleted for Dcp2 and Xrn1 using the AID system. Top panel shows the quantification of the autoradiogram. H . Graph shows the relative number of mRNAs with median pA-tails clustered in bins of 5 for transcripts represented by at least 30 reads for control compared Dcp2- and Xrn1-depleted cells. I-J . Scatterplots show the change in mRNA abundance on the x-axis in relation to the absolute change in mean pA-tail length for (I) Dcp2- and (J) Xrn1-depleted cells. K . Scatterplot compares the absolute change in mean pA-tail length in Dcp2- and Xrn1-depleted cells. L . Scatterplot shows the absolute change in mean pA-tail length in relation to the change in mRNA abundance between a wild-type sample digested or not with Xrn1 in vitro . M-N . Graphs show pA-tail length distributions of HHF1 mRNA in (M) control cells in comparison to wild-type sample digested in vitro with Xrn1 and in pop2Δ and for (N) control cells in comparison to Dcp2 and Xrn1 depletion using AID. O-P . Same as (M-N.) for RPS13 mRNA. R . Scatterplot shows the absolute change in mean pA-tail length for wild-type cells treated with Xrn1 in vitro compared to control and pop2Δ cells in relation to the same control sample. S . Scatterplot shows the absolute change in mean pA-tail length for ccr4Δ and pop2Δ cells compared to the same wild-type sample.

    Article Snippet: Purified Tt Xrn1 was inspected in 10% SDS-PAGE stained with Coomassie Brilliant Blue R-250, along with commercial yeast Xrn1 (NEB; M0338) as a positive control (Supplementary Figure X).

    Techniques: Western Blot, Produced, In Vitro

    ( A ) Digestion of 2′-phosphorylated RNA by Xrn1 yields a product that inhibits further degradation. ( B ) The 2′-phosphate on tRNA-Pro-UGG can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . In the lower panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. Asterisks correspond to previously described but uncharacterized Pro-TGG splicing intermediates , . ( C ) Unmodified, 2′-phosphorylated, and 2′-O-methylated RNA substrates show zero, substantial, and trace resistance to Xrn1 digestion, respectively. ( D ) A 2′-phosphate inhibits decay by the 5′→3′ exonucleases Xrn1, Dxo1, and RNase J1. The location of the 2′-phosphorylated linkage is indicated by the red “G” in the substrate at right, with inferred stalling sites for each exonuclease marked with arrows. ( E ) In budding yeast, the unfolded protein response (UPR) initiates a noncanonical splicing event on the mRNA HAC1 during which the endonuclease Ire1 excises the HAC1 intron (thin line), followed by exon ligation by Trl1 (black and gray boxes), generating an internal 2′-phosphate at the splice junction that is subsequently removed by the 2′-phosphotransferase Tpt1. ( F ) RNA from WT and xrn1Δ tpt1Δ (10x-tRNA) cells was treated with DMSO or tunicamycin and left untreated (left panel) or enzymatically and digested (right panel) followed by nanopore sequencing. 5′-end alignments of reads are expressed in counts per million reads (CPM). ( G ) Subtraction of undigested background signal from signal from the Tm-treated, rXrn1-digested libraries in ( F ), yields a predominant 5′-end signal on HAC1 s at the splice junction (yellow box). At right, an enhanced view of this region shows the major peak in wild-type cells is located precisely at the exon-exon junction (position 727), whereas the end signal in the mutant is 13 nucleotides downstream, an offset consistent with premature termination of base calling when a bona fide 5′ RNA end exits a nanopore. ( H ) The wild-type rXrn1 enrichment scores above have been subtracted from the mutant scores at each nucleotide position.

    Journal: bioRxiv

    Article Title: Direct detection of RNA repair by nanopore sequencing

    doi: 10.1101/2022.05.29.493267

    Figure Lengend Snippet: ( A ) Digestion of 2′-phosphorylated RNA by Xrn1 yields a product that inhibits further degradation. ( B ) The 2′-phosphate on tRNA-Pro-UGG can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . In the lower panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. Asterisks correspond to previously described but uncharacterized Pro-TGG splicing intermediates , . ( C ) Unmodified, 2′-phosphorylated, and 2′-O-methylated RNA substrates show zero, substantial, and trace resistance to Xrn1 digestion, respectively. ( D ) A 2′-phosphate inhibits decay by the 5′→3′ exonucleases Xrn1, Dxo1, and RNase J1. The location of the 2′-phosphorylated linkage is indicated by the red “G” in the substrate at right, with inferred stalling sites for each exonuclease marked with arrows. ( E ) In budding yeast, the unfolded protein response (UPR) initiates a noncanonical splicing event on the mRNA HAC1 during which the endonuclease Ire1 excises the HAC1 intron (thin line), followed by exon ligation by Trl1 (black and gray boxes), generating an internal 2′-phosphate at the splice junction that is subsequently removed by the 2′-phosphotransferase Tpt1. ( F ) RNA from WT and xrn1Δ tpt1Δ (10x-tRNA) cells was treated with DMSO or tunicamycin and left untreated (left panel) or enzymatically and digested (right panel) followed by nanopore sequencing. 5′-end alignments of reads are expressed in counts per million reads (CPM). ( G ) Subtraction of undigested background signal from signal from the Tm-treated, rXrn1-digested libraries in ( F ), yields a predominant 5′-end signal on HAC1 s at the splice junction (yellow box). At right, an enhanced view of this region shows the major peak in wild-type cells is located precisely at the exon-exon junction (position 727), whereas the end signal in the mutant is 13 nucleotides downstream, an offset consistent with premature termination of base calling when a bona fide 5′ RNA end exits a nanopore. ( H ) The wild-type rXrn1 enrichment scores above have been subtracted from the mutant scores at each nucleotide position.

    Article Snippet: To prepare exonuclease-degraded mRNA, 200 µg of total RNA was decapped with mRNA decapping enzyme (New England Biolabs) for 1 hour at 37 ºC, ethanol precipitated, resuspended, and split into two 20 µL reactions in the buffer described above, with or without 2 µL of recombinant Xrn1 (1.1 mg/mL).

    Techniques: Northern Blot, Recombinant, In Vitro, Methylation, Ligation, Nanopore Sequencing, Mutagenesis

    ( A ) Unmodified 20 nt oligo and 2′-phosphorylated 26 nt oligo used in this experiment. ( B ) 26 nt oligos were 3’-end labeled with radiolabeled pCp prior to incubation with or without rXrn1. After digestion, all samples were treated with phenol:chloroform and ethanol precipitated, and then mixed with equimolar amounts of the 20 nt oligo, followed by a 1 hour incubation with or without T4 RNL1. Ligation products are visible in lane 2, but undetectable in lane 4.

    Journal: bioRxiv

    Article Title: Direct detection of RNA repair by nanopore sequencing

    doi: 10.1101/2022.05.29.493267

    Figure Lengend Snippet: ( A ) Unmodified 20 nt oligo and 2′-phosphorylated 26 nt oligo used in this experiment. ( B ) 26 nt oligos were 3’-end labeled with radiolabeled pCp prior to incubation with or without rXrn1. After digestion, all samples were treated with phenol:chloroform and ethanol precipitated, and then mixed with equimolar amounts of the 20 nt oligo, followed by a 1 hour incubation with or without T4 RNL1. Ligation products are visible in lane 2, but undetectable in lane 4.

    Article Snippet: To prepare exonuclease-degraded mRNA, 200 µg of total RNA was decapped with mRNA decapping enzyme (New England Biolabs) for 1 hour at 37 ºC, ethanol precipitated, resuspended, and split into two 20 µL reactions in the buffer described above, with or without 2 µL of recombinant Xrn1 (1.1 mg/mL).

    Techniques: Labeling, Incubation, Ligation

    ( A ) Cloverleaf diagram of a spliced tRNA from tpt1Δ cells, indicating the 2′-phosphorylated nucleotide located immediately 3′ of the anticodon, and the 5mer centered on this position in gray. ( B ) The RNA modification landscape within the indicated 5mer in ( A ) for each intron-containing tRNA in S. cerevisiae . Blue boxes indicate positions with known tRNA modifications in the MODOMICS database. ( C ) The 2′-phosphate on tRNA-Trp-CCA can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . 3′-exon degradation intermediates accumulate in tpt1Δ (10x-tRNA) cells, while two larger exonuclease-resistant bands are also present, albeit less abundant, in RNA from trl1Δ (10x-tRNA) cells treated with rXrn1. Given that the MODOMICS database lists annotated methyl groups on this mature tRNA at nucleotides 31 and 33 , and that 2′-O-methyl groups also inhibit Xrn1 , albeit substantially less robustly than 2′-phosphates , it is possible that these larger bands are produced by 2′-O-methyl-mediated rXrn1 inhibition. However, in the context of a trl1Δ tpt1Δ double mutant, these degradation intermediates disappear, raising the possibility of additional crosstalk between tRNA splicing and tRNA modification pathways. In the lower sub-panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. ( D ) IGV snapshots of direct tRNA sequencing reads mapping to intron-containing S. cerevisiae tRNAs. Each colored bar represents a position with >20% mismatching to the reference base; gray bars indicate positions which did not exceed this threshold. Numbers in brackets at right indicate the Y axis range in read counts, and tRNA anticodons are surrounded by a black box. Modified positions reported in MODOMICS are annotated below the reference sequence. To the right of select IGV alignment views are northern blots of total RNA from S. cerevisiae strains treated with and without recombinant Xrn1 in vitro , using a 3′-exon hybridizing probe.

    Journal: bioRxiv

    Article Title: Direct detection of RNA repair by nanopore sequencing

    doi: 10.1101/2022.05.29.493267

    Figure Lengend Snippet: ( A ) Cloverleaf diagram of a spliced tRNA from tpt1Δ cells, indicating the 2′-phosphorylated nucleotide located immediately 3′ of the anticodon, and the 5mer centered on this position in gray. ( B ) The RNA modification landscape within the indicated 5mer in ( A ) for each intron-containing tRNA in S. cerevisiae . Blue boxes indicate positions with known tRNA modifications in the MODOMICS database. ( C ) The 2′-phosphate on tRNA-Trp-CCA can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro . 3′-exon degradation intermediates accumulate in tpt1Δ (10x-tRNA) cells, while two larger exonuclease-resistant bands are also present, albeit less abundant, in RNA from trl1Δ (10x-tRNA) cells treated with rXrn1. Given that the MODOMICS database lists annotated methyl groups on this mature tRNA at nucleotides 31 and 33 , and that 2′-O-methyl groups also inhibit Xrn1 , albeit substantially less robustly than 2′-phosphates , it is possible that these larger bands are produced by 2′-O-methyl-mediated rXrn1 inhibition. However, in the context of a trl1Δ tpt1Δ double mutant, these degradation intermediates disappear, raising the possibility of additional crosstalk between tRNA splicing and tRNA modification pathways. In the lower sub-panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. ( D ) IGV snapshots of direct tRNA sequencing reads mapping to intron-containing S. cerevisiae tRNAs. Each colored bar represents a position with >20% mismatching to the reference base; gray bars indicate positions which did not exceed this threshold. Numbers in brackets at right indicate the Y axis range in read counts, and tRNA anticodons are surrounded by a black box. Modified positions reported in MODOMICS are annotated below the reference sequence. To the right of select IGV alignment views are northern blots of total RNA from S. cerevisiae strains treated with and without recombinant Xrn1 in vitro , using a 3′-exon hybridizing probe.

    Article Snippet: To prepare exonuclease-degraded mRNA, 200 µg of total RNA was decapped with mRNA decapping enzyme (New England Biolabs) for 1 hour at 37 ºC, ethanol precipitated, resuspended, and split into two 20 µL reactions in the buffer described above, with or without 2 µL of recombinant Xrn1 (1.1 mg/mL).

    Techniques: Modification, Northern Blot, Recombinant, In Vitro, Produced, Inhibition, Mutagenesis, Sequencing