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The exonuclease torpedo Xrn2 loads onto nascent RNA 5′-PO4ends and chases down pol II to promote termination downstream from polyA sites. We report that Xrn2 is recruited to preinitiation complexes and “travels” to 3′ ends of genes. Mapping of 5′-PO4ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are approximately two to 20 bases downstream from where CPSF73 cleaves at polyA sites and histone 3′ ends. We propose that processing of all mRNA 3′ ends comprises cleavage and limited 5′–3′ trimming by CPSF73, followed by handoff to Xrn2. A similar handoff occurs at tRNA 3′ ends, where cotranscriptional RNase Z cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3′ flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3′ flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and antisense nascent RNA within a few bases of the TSS, where 5′-PO4ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminates prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.

Alternative polyadenylation yields many mRNA isoforms whose 3’ termini occur disproportionately in clusters within 3’ untranslated regions. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et al., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. Within yeast isoform clusters, these shifts occur steadily from one isoform to the next across nucleotide distances. In contrast, the shift between clusters – from the last isoform of one cluster to the first isoform of the next – is much less pronounced, even over large distances. GC content in a region 13–30 nt downstream from isoform clusters correlates with their sensitivity to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at single nucleotide resolution within clusters but not between them. Pol II occupancy increases just downstream of poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that (1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, (2) poly(A) site clusters are linked to the local elongation rate, and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, (3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and (4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

Alternative polyadenylation yields many mRNA isoforms whose 3’ termini occur disproportionately in clusters within 3’ UTRs. Previously, we showed that profiles of poly(A) site usage are regulated by the rate of transcriptional elongation by RNA polymerase (Pol) II (Geisberg et., 2020). Pol II derivatives with slow elongation rates confer an upstream-shifted poly(A) profile, whereas fast Pol II strains confer a downstream-shifted poly(A) profile. In yeast, upstream and downstream shifts within isoform clusters occur steadily at the nucleotide level. In contrast, changes from one isoform to the next are much smaller between clusters, even when the distances between them are relatively large. GC content in a region 13-30 nt downstream from isoform clusters is linked to Pol II elongation rate. In human cells, the upstream shift caused by a slow Pol II mutant also occurs continuously at the nucleotide level within clusters, but not between them. Pol II occupancy increases just downstream of the most speed-sensitive poly(A) sites, suggesting a linkage between reduced elongation rate and cluster formation. These observations suggest that 1) Pol II elongation speed affects the nucleotide-level dwell time allowing polyadenylation to occur, 2) poly(A) site clusters are linked to the local elongation rate and hence do not arise simply by intrinsically imprecise cleavage and polyadenylation of the RNA substrate, 3) DNA sequence elements can affect Pol II elongation and poly(A) profiles, and 4) the cleavage/polyadenylation and Pol II elongation complexes are spatially, and perhaps physically, coupled so that polyadenylation occurs rapidly upon emergence of the nascent RNA from the Pol II elongation complex.

Background The sequence content of the 3′ UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging. Results To combat this problem, we developed LABRAT, an APA isoform quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA isoform abundance in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between relative APA isoform abundance and overall gene expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA isoforms, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated. Conclusions We conclude that LABRAT has the ability to accurately quantify APA isoform ratios from RNAseq data across a variety of sample types. Further, LABRAT is able to derive biologically meaningful insights that connect APA isoform regulation to cellular and molecular phenotypes.

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from2 kb/min to 1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

Summary In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3′ flanking regions where termination is enhanced by TFIISDN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5′ pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions.

CDK7 phosphorylates the RNA polymerase II (pol II) CTD and activates the P-TEFb-associated kinase, CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoterproximal pausing, and defective termination at gene 3'-ends upon CDK7 inhibition. Surprisingly, we also noted CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5'-ends and H3K36me3 was displaced toward gene 3'-ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated vs. P-TEFb-phosphorylated CTD (vs. unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7 dependent "CTD code" that regulates epigenetic marks in addition to RNA processing and pol II pausing.

The torpedo model of transcription termination asserts that the exonuclease Xrn2 attacks the 5'PO4-end exposed by nascent RNA cleavage and chases down the RNA polymerase. We tested this mechanism using a dominant-negative human Xrn2 mutant and found that it delayed termination genome-wide. Xrn2 nuclease inactivation caused strong termination defects downstream of most poly(A) sites and modest delays at some histone and U snRNA genes, suggesting that the torpedo mechanism is not limited to poly(A) site-dependent termination. A central untested feature of the torpedo model is that there is kinetic competition between the exonuclease and the pol II elongation complex. Using pol II rate mutants, we found that slow transcription robustly shifts termination upstream, and fast elongation extends the zone of termination further downstream. These results suggest that kinetic competition between elongating pol II and the Xrn2 exonuclease is integral to termination of transcription on most human genes.

Transcription elongation rate influences cotranscriptional pre-mRNA maturation, but how such kinetic coupling works is poorly understood. The formation of nonadenylated histone mRNA 3′ ends requires recognition of an RNA structure by stem–loop-binding protein (SLBP). We report that slow transcription by mutant RNA polymerase II (Pol II) caused accumulation of polyadenylated histone mRNAs that extend past the stem–loop processing site. UV irradiation, which decelerates Pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3′ processing by slow Pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem–loop fails to fold in transcripts made by slow Pol II, thereby explaining the absence of SLBP and failure to process 3′ ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure and suggest a mechanism by which alternative processing could be controlled.

Summary: CDK7 phosphorylates the RNA polymerase II (pol II) C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3′ ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5′ ends and H3K36me3 was displaced toward gene 3′ ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylated CTD stimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent “CTD code” that regulates chromatin marks in addition to RNA processing and pol II pausing. : Ebmeier et al. use targeted proteomics to identify distinct interactomes for full-length mammalian pol II CTD phosphorylated by TFIIH or P-TEFb. In vitro and cell-based assays uncover expanded roles for the TFIIH-associated kinase CDK7, including regulation of SETD1A/B methyltransferase activity and regulation of transcription-associated histone modifications (i.e., H3K4me3 and H3K36me3). Keywords: RNA-seq, proteomics, Mediator, THZ1, chromatin, epigenetic, TFIIH, P-TEFb, H3K4me3, H3K36me3

A chromatin code appears to mark introns and exons with distinct patterns of nucleosome enrichment and histone methylation. We investigated whether a causal relationship exists between splicing and chromatin modification by asking whether splice-site mutations affect the methylation of histone H3K36. Deletions of the 3′ splice site in intron 2 or in both introns 1 and 2 of an integrated β-globin reporter gene caused a shift in relative distribution of H3K36 trimethylation away from 5′ ends and toward 3′ ends. The effects of splice-site mutations correlated with enhanced retention of a U5 snRNP subunit on transcription complexes downstream of the gene. In contrast, a poly(A) site mutation did not affect H3K36 methylation. Similarly, global inhibition of splicing by spliceostatin A caused a rapid repositioning of H3K36me3 away from 5′ ends in favor of 3′ ends. These results suggest that the cotranscriptional splicing apparatus influences establishment of normal patterns of histone modification.

In this study, Saldi et al. investigated how transcription elongation rate influences cotranscriptional pre-mRNA maturation. Their findings show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure and suggest a mechanism by which alternative processing could be controlled.

The C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) influences many steps in the synthesis of an mRNA and helps control the final destiny of the mature transcript. ADAR2 edits RNA by converting adenosine to inosine within double-stranded or structured RNA. Site-selective A-to-I editing often occurs at sites near exon/intron borders, where it depends on intronic sequences for substrate recognition. It is therefore essential that editing precedes splicing. We have investigated whether there is coordination between ADAR2 editing and splicing of the GluR-B pre-mRNA. We show that the CTD is required for efficient editing at the R/G site one base upstream of a 5′-splice site. The results suggest that the CTD enhances editing at the R/G site by preventing premature splicing that would remove the intronic recognition sites for ADAR2. Editing at the GluR-B Q/R site, 24 bases upstream of the intron 11 5′-splice site, stimulates splicing at this intron. Furthermore, unlike previously studied introns, the CTD actually inhibits excision of intron 11, which includes the complementary recognition sequences for the Q/R editing site. In summary, these results show that the CTD and ADAR2 function together to enforce the order of events that allows editing to precede splicing, and they furthermore suggest a new role for the CTD as a coordinator of two interdependent pre-mRNA processing events.

The cellular transcription and mRNA processing machineries appear to have co-evolved to allow coupling of the reactions they perform in space and time. Coupling in space is achieved by “recruitment” of processing factors to the nascent transcript, often by binding to the pol II CTD. Much less is known about how coupling in time is achieved, that is to say the coordination of rates of elongation and processing. Slow elongation is predicted to lengthen the window of opportunity for an event to occur at an upstream site on the nascent transcript before it must compete with an RNA sequence element further downstream. Consistent with this prediction, in a few cases slow elongation is associated with enhanced inclusion of alternative exons (ref. 1) through processing at upstream splice sites and processing at upstream alternative poly(A) sites (ref. 2). We investigated kinetic coupling in human cell lines that express pol II large subunits with mutations in funnel and trigger loop domains that slow down and spe...

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (pol II) is essential for several co-transcriptional pre-messenger RNA processing events, including capping, 3′-end processing and splicing. We investigated the role of the CTD of RNA pol II in the coordination of A to I editing and splicing of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre-mRNA. The auto-editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3′ splice site. Unlike other introns, the CTD is not required for efficient splicing of intron 4 at either the normal 3′ splice site or the alternative site created by editing. However, the CTD is required for efficient co-transcriptional auto-editing of ADAR2 intron 4. Our results implicate the CTD in site-selective RNA editing by ADAR2 and in coordination of editing with alternative splicing.

Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription.

Alternative splicing modulates expression of most human genes. The kinetic model of cotranscriptional splicing suggests that slow elongation expands and that fast elongation compresses the “window of opportunity” for recognition of upstream splice sites, thereby increasing or decreasing inclusion of alternative exons. We tested the model using RNA polymerase II mutants that change average elongation rates genome-wide. Slow and fast elongation affected constitutive and alternative splicing, frequently altering exon inclusion and intron retention in ways not predicted by the model. Cassette exons included by slow and excluded by fast elongation (type I) have weaker splice sites, shorter flanking introns, and distinct sequence motifs relative to “slow-excluded” and “fast-included” exons (type II). Many rate-sensitive exons are misspliced in tumors. Unexpectedly, slow and fast elongation often both increased or both decreased inclusion of a particular exon or retained intron. These results suggest that an optimal rate of transcriptional elongation is required for normal cotranscriptional pre-mRNA splicing.

Capping, splicing, and cleavage/polyadenylation of pre-mRNAs are interdependent events that are all stimulated in vivo by the carboxy-terminal domain (CTD) of RNA Pol II. We show that the CTD independently enhances splicing and 3′ processing and that stimulation of splicing by enhancers is facilitated by the CTD. We provide evidence that stimulation of 3′ processing by the CTD requires contact with the 50-kD subunit of the cleavage stimulation factor, CstF. Overexpression of the CTD-binding domain of CstF p50 had a dominant-negative effect on 3′ processing without disrupting the CstF complex. The CTD comprises 52 heptad repeats. The CTD carboxyl terminus including heptads 27–52 supported capping, splicing, and 3′ processing but the amino terminus supported only capping. We conclude that the CTD independently stimulates all three major pre-mRNA processing steps and that different regions of the CTD can serve distinct functions in pre-mRNA processing.

Summary We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a, and Dcp2 and the termination factor TTF2 coimmunoprecipitate with Xrn2, the nuclear 5′–3′ exonuclease "torpedo" that facilitates transcription termination at the 3′ ends of genes. Dcp1a, Xrn2, and TTF2 localize near transcription start sites (TSSs) by ChIP-seq. At genes with 5′ peaks of paused pol II, knockdown of decapping or termination factors Xrn2 and TTF2 shifted polymerase away from the TSS toward upstream and downstream distal positions. This redistribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the "torpedo" mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated cotranscriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.

We report that under 'transcriptional stress' in budding yeast, when most pol II activity is acutely inhibited, rapid deposition of nucleosomes occurs within genes, particularly at 3' positions. Whereas histone H3K4 trimethylation normally marks 5' ends of highly transcribed genes, under 'transcriptional stress' induced by 6-azauracil (6-AU) and inactivation of pol II, TFIIE or CTD kinases Kin28 and Ctk1, this mark shifted to the 3' end of the TEF1 gene. H3K4Me3 at 3' positions was dynamic and could be rapidly removed when transcription recovered. Set1 and Chd1 are required for H3K4 trimethylation at 3' positions when transcription is inhibited by 6-AU. Furthermore, Deltachd1 suppressed the growth defect of Deltaset1. We suggest that a 'transcriptional stress' signal sensed through Set1, Chd1, and possibly other factors, causes H3K4 hypermethylation of newly deposited nucleosomes at downstream positions within a gene. This response identifies a new role for H3K4 trimethylation at the 3' end of the gene, as a chromatin mark associated with impaired pol II transcription.

The RNA polymerase II C-terminal heptad repeat domain (CTD) is essential for normal transcription and co-transcriptional processing of mRNA precursors. The mammalian CTD comprises 52 heptads whose consensus, YSPTSPS, is conserved throughout eukaryotes, followed by a 10 amino acid C-terminal sequence that is conserved only among vertebrates. Here we show that surprisingly, the heptad repeats are not sufficient to support efficient transcription, pre-mRNA processing or full cell viability. In addition to the heptads, the 10 amino acid C-terminal motif is essential for high level transcription, splicing and poly(A) site cleavage. Efficient mRNA synthesis from a transiently transfected reporter gene required the C-terminal motif plus between 16 and 25 heptad repeats from either the N- or C-terminal half of the CTD. Twenty-seven consensus heptads plus the C-terminal motif also supported efficient mRNA synthesis but not cell viability.

We have investigated the role of the RNA Polymerase II (Pol II) carboxy-terminal domain (CTD) in mRNA 5′ capping. Transcripts made in vivo by Pol II with a truncated CTD had a lower proportion of capped 5′ ends than those made by Pol II with a full-length CTD. In addition, the enzymes responsible for cap synthesis, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase bound directly to the phosphorylated, but not to the nonphosphorylated, form of the CTD in vitro. These results suggest that: (1) Pol II-specific capping of nascent transcripts in vivo is enhanced by recruitment of the capping enzymes to the CTD and (2) capping is co-ordinated with CTD phosphorylation.

Messenger RNA is produced by RNA polymerase II (pol II) transcription, followed by processing of the primary transcript. Transcription, splicing and cleavage-polyadenylation can occur independently in vitro, but we demonstrate here that these processes are intimately linked in vivo. We show that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient RNA processing. Splicing, processing of the 3' end and termination of transcription downstream of the poly(A) site, are all inhibited by truncation of the CTD. We found that the cleavage-polyadenylation factors CPSF and CstF specifically bound to CTD affinity columns and copurified with pol II in a high-molecular-mass complex. Our demonstration of an association between the CTD and 3'-processing factors, considered together with reports of a similar interaction with splicing factors, suggests that an mRNA 'factory' exists which carries out coupled transcription, splicing and cleavage-polyadenylation of mRNA precursors.