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

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

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

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

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

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

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