The Exosome Component Rrp6 Is Required for RNA Polymerase II Termination at Specific Targets of the Nrd1-Nab3 Pathway

Publisher’s version made available under a Creative Commons license.The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3' end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3' end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes

The interactome of the atypical phosphatase Rtr1 in Saccharomyces cerevisiae

The phosphatase Rtr1 has been implicated in dephosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) during transcription elongation and in regulation of nuclear import of RNAPII. Although it has been shown that Rtr1 interacts with RNAPII in yeast and humans, the specific mechanisms that underlie Rtr1 recruitment to RNAPII have not been elucidated. To address this, we have performed an in-depth proteomic analysis of Rtr1 interacting proteins in yeast. Our studies revealed that hyperphosphorylated RNAPII is the primary interacting partner for Rtr1. To extend these findings, we performed quantitative proteomic analyses of Rtr1 interactions in yeast strains deleted for CTK1, the gene encoding the catalytic subunit of the CTD kinase I (CTDK-I) complex. Interestingly, we found that the interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains. We hypothesize that serine-2 CTD phosphorylation is required for Rtr1 recruitment to RNAPII during transcription elongation

RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome

Rrp6 is required for NNS-dependent termination and RNA processing of the snR71 transcript.

<p>(A) Graphical representation of strand-specific RNA-seq reads mapped to snR71-extended region. Reads mapped to the positive strand are on top in red, while reads mapped to the negative strand are on the bottom in blue. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Processed length of snRNAs and mRNAs are in black, snRNA-<u>e</u>xtended <u>t</u>ranscripts, including pre-snRNAs and termination read-through products, are in green (labeled “ETs”), NUTs are in aqua, CUTs are in gray, and bent arrows indicate direction of transcription. The dotted black line marks the transcription start site (TSS) of <i>LIN1</i>. (B) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the same region and aligned to (A). Wild-type normalized read counts are in black, and <i>rrp6Δ</i> are in orange. Red arrows indicate areas of discussion in the text. (C) Average normalized read counts ± standard deviations for significantly altered transcripts in <i>rrp6Δ</i> versus wild-type (n = 4). One star indicates a p-value of <0.05, two stars indicate a p-value of <0.01, and three stars indicate a p-value of <0.001 as determined by an unpaired, two-tailed student’s t-test. The colors of the bars in each graph correspond to the color representing the related annotation. (D) Strand-specific northern blot analysis using a 5’ end labeled probe specific to the processed region of snR71 directly comparing <i>rrp6Δ</i> to NNS termination mutants. The 26S and 18S ribosomal RNAs are shown as a loading control (bottom).</p

Expression plots for normalized RNA-SEQ data with specific classes of RNA transcripts highlighted.

<p>After sequencing reads were aligned to the yeast genome, reads mapped to annotated open reading frame transcripts (ORF-Ts), cryptic unstable transcripts (CUTs), stable unannotated transcripts (SUTs), and Nrd1 unterminated transcripts (NUTs) were used for differential expression analysis using edgeR. Log₂ of the fold-change values are plotted versus the average number of normalized reads across all biological replicates for all RNA transcripts in cells lacking <i>RRP6</i> compared to WT (black dots). (A) RNAs annotated as CUTs, a classification based on the dependence of <i>rrp6Δ</i> for detection are shown as gray dots while all other transcripts are shown as black dots. (B) RNAs annotated as ORF-Ts, most of which are protein coding messenger RNAs are shown in yellow. All other transcripts are shown in black. (C) Messenger RNA expression values for ribosomal protein coding genes, shown as black dots.</p

Comparison of highly abundant sn/snoRNA transcripts from <i>rrp6Δ</i> and WT strains obtained through tiling array or RNA sequencing.

<p>(A) Comparison of a recent <i>rrp6Δ</i> tiling array dataset [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004999#pgen.1004999.ref051" target="_blank">51</a>] and RNA-sequencing data collected in this study by plotting the average log₂ ratio values (<i>rrp6Δ</i> / WT) for all transcripts. All annotated transcripts included in both data sets are represented by gray circles. All sn/snoRNAs are highlighted in red circles. The Pearson correlation coefficient (<i>r</i>) between these datasets = 0.751, indicating a modest positive correlation between the two data sets. (B) Area under the curve calculation for snR37 extended transcripts (labeled as “ET”) from wild-type and <i>rrp6Δ</i> strains. The area calculated for snR37-ET annotation was divided by the area calculated for the entire snR37 to snR37-ET annotation to calculate the percentage of the entire transcript area located in the snR37-ET region (see diagram under (C) and (D) for locations of annotations). Values are shown as averages ± standard deviations for sequencing data are in black, tiling array data is in red. (C) Previously published tiling array intensity values [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004999#pgen.1004999.ref059" target="_blank">59</a>] at snR37 using probe mid-position (8 nucleotides apart), for comparison with (D) mapped reads from RNA-Seq data at snR37 (single nucleotide resolution). Tiling array data represented in log₂ scale. RNA-Seq data represented in log₁₀ scale. In both graphs, wild-type RNA levels are shown in black, and <i>rrp6Δ</i> RNA levels are in red. Locations of annotated snRNA transcript (shown in black) and the extended transcript region annotated in this study (shown in green) are drawn to scale on each plot.</p

Termination of the C/D box small nucleolar RNA snR13 transcript does not require Rrp6.

<p>(A) Graphical representation of strand-specific RNA-seq reads mapped to snR13-<i>YDR473C</i> region. Reads mapped to the positive strand are on top in red, while reads mapped to the negative strand are on the bottom in blue. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Processed length of snRNAs and mRNAs are in black, snRNA-<u>e</u>xtended <u>t</u>ranscripts, including pre-snRNAs and termination read-through products, are in green (labeled “ETs”), NUTs are in aqua, and arrows indicate direction of transcription. The dotted black line marks the transcription start site (TSS) of <i>TRS31</i>. (B) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the same region and aligned to (A). Wild-type normalized read counts are in black, and <i>rrp6Δ</i> are in orange. Nrd1 binding sites as determined by PAR-CLIP from Creamer <i>et al</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004999#pgen.1004999.ref039" target="_blank">39</a>] are shown for comparison in aqua. (C) Average normalized read counts ± standard deviations for significantly altered transcripts in <i>rrp6Δ</i> versus wild-type (n = 4). Two stars represent a p-value of <0.01 as determined by an unpaired, two-tailed student’s t-test. The colors of the bars in each graph correspond to the color representing the related annotation. (D) Strand-specific northern blot analysis using a 5’ end labeled DNA oligonucleotide probe specific to the processed region of snR13 directly comparing <i>rrp6Δ</i> to mutants known to be defective in Nrd1-dependent termination. The temperature sensitive <i>nrd1Δ151–214</i> strain was grown at 30°C overnight, diluted to an OD₆₀₀ of 0.5, and grown at 37°C for 30 minutes or 60 minutes as indicated. The <i>ssu72 TOV</i> strain has been previously shown by Loya et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004999#pgen.1004999.ref060" target="_blank">60</a>] to bypass Nrd1-dependent termination at the <i>IMD2</i> locus. The 26S and 18S ribosomal RNAs are shown as a loading control (bottom).</p

<i>NRD1</i> early termination requires Rrp6 for efficient RNAPII termination.

<p>(A) Early termination model of NNS-dependent transcription attenuation. A hypothetical gene ‘Gene X’ is shown with the coding region (solid lines) and UTRs (dashed lines) drawn. Locations of Nrd1 or Nab3 binding sites (aqua) or the Polyadenylation site (red) are drawn above the figure. In brief, early termination by the NNS pathway prevents transcription of the full length gene. (B) Average normalized read counts ± standard deviations for <i>NRD1</i> and CUT320 transcripts in <i>rrp6Δ</i> versus wild-type (n = 3). One star indicates a p-value of <0.05 and two stars indicate a p-value of <0.01. (C) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the <i>NRD1</i> region. Wild-type reads are in black, and <i>rrp6Δ</i> are in orange. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Length of mRNAs including untranslated regions are in black, CUTs are in gray, and NUTs are in aqua. Nrd1 binding sites as determined by PAR-CLIP from Creamer <i>et al</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004999#pgen.1004999.ref039" target="_blank">39</a>] are shown in aqua for comparison. The scale for Nrd1 PAR-CLIP data is shown to the right.</p

The <i>FMP40</i> antisense transcript <i>YPL222C-A</i> is extended in <i>rrp6Δ</i> deletion cells as a result of inefficient RNAPII termination.

<p>(A) Graphical representation of strand-specific RNA-seq reads mapped to <i>FMP40</i> region. Reads mapped to the positive strand are on top in red, while reads mapped to the negative strand are on the bottom in blue. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Length of mRNAs including untranslated regions are in black, CUTs are in gray, dubious ORFs (an antisense transcript named <i>YPL222C-A</i>) are in tan, and bent arrows indicate direction of the TSS. The dotted black lines mark the 3’ end of the annotations for CUT882 and <i>YPL222C-A</i>. (B) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the same region and aligned to (A). Wild-type reads are in black, and <i>rrp6Δ</i> are in orange. Red arrows note areas of interest. (C) Average normalized read counts ± standard deviations for significantly altered transcripts in <i>rrp6Δ</i> versus wild-type (n = 4). Three stars indicate a p-value of <0.001 as determined by an unpaired, two-tailed student’s t-test. The colors of the bars in each graph correspond to the color representing the related annotation. (D) Strand-specific northern blot analysis using a 5’ end labeled probes specific to either <i>FMP40</i> or <i>YPL222C-A</i> directly comparing <i>rrp6Δ</i> to mutants known to be defective in NNS termination. The 26S and 18S ribosomal RNAs are shown as a loading control (bottom).</p