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• W2897066913 abstract "•S5P CTD Pol II associates with the catalytic spliceosome•Elongating Pol II complexes protect about 60 newly synthesized nucleotides•Co-transcriptional splicing associated with dominant 5′ ss intermediate•U12-dependent introns are sequentially spliced in association with Pol II The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5′ splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3′ splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion. The highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5′ splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3′ splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion. Eukaryotic protein coding genes often contain multiple introns that are removed from primary transcripts of RNA polymerase II (Pol II) by splicing. This is catalyzed by the spliceosome, comprising five snRNAs and more than 200 proteins, a subset of which form functional ribonucleoprotein particles. Exon-intron boundaries are defined by short consensus sequences at the 5′ and 3′ splice sites (ss) that mediate recognition by the spliceosome. Spliceosome assembly also requires recognition of an intronic catalytic adenosine (branch point) and, for mammalian introns, a polypyrimidine tract located between the branch point adenosine and 3′ ss. Within the 3D structure of the spliceosome, these key pre-mRNA sequences are forced into an RNA catalytic center that triggers splicing (Papasaikas and Valcárcel, 2016Papasaikas P. Valcárcel J. The Spliceosome: The Ultimate RNA Chaperone and Sculptor.Trends Biochem. Sci. 2016; 41: 33-45Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). This involves two transesterification reactions. First, the 2′ hydroxyl group (OH) of the branch point adenosine carries out a nucleophilic attack on the phosphate group at the 5′ ss; then the exposed 3′ OH of the upstream exon cleaves the lariat structured intron away from the 3′ ss, resulting in covalent joining of the two exons (Patel and Steitz, 2003Patel A.A. Steitz J.A. Splicing double: insights from the second spliceosome.Nat. Rev. Mol. Cell Biol. 2003; 4: 960-970Crossref PubMed Scopus (311) Google Scholar). Exactly how two splice sites separated by extensive intronic sequence (in mammals, 1 to 100 kb) are brought together during early stages of spliceosome assembly remains a puzzle. Possibly, the 5′ exon is tethered to elongating Pol II until complete synthesis of the downstream intron has occurred. This would maintain the two splice sites in close proximity, irrespective of intron size (Hollander et al., 2016Hollander D. Naftelberg S. Lev-Maor G. Kornblihtt A.R. Ast G. How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing?.Trends Genet. 2016; 32: 596-606Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Co-transcriptional tethering of the 5′ exon may involve the flexible C-terminal domain (CTD) of Pol II’s largest subunit, Rbp1, which acts to coordinate transcription with pre-mRNA processing (Heidemann et al., 2013Heidemann M. Hintermair C. Voß K. Eick D. Dynamic phosphorylation patterns of RNA polymerase II CTD during transcription.Biochim. Biophys. Acta. 2013; 1829: 55-62Crossref PubMed Scopus (182) Google Scholar, Zaborowska et al., 2016Zaborowska J. Egloff S. Murphy S. The pol II CTD: new twists in the tail.Nat. Struct. Mol. Biol. 2016; 23: 771-777Crossref PubMed Scopus (133) Google Scholar). Mammalian CTD comprises 52 repeats of a heptad with the consensus amino acid sequence YSPTSPS. This is extensively modified post-translationally, principally by phosphorylation to S2P, S5P, S7P, T4P, and Y1P (Hintermair et al., 2012Hintermair C. Heidemann M. Koch F. Descostes N. Gut M. Gut I. Fenouil R. Ferrier P. Flatley A. Kremmer E. et al.Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation.EMBO J. 2012; 31: 2784-2797Crossref PubMed Scopus (108) Google Scholar, Hsin and Manley, 2012Hsin J.P. Manley J.L. The RNA polymerase II CTD coordinates transcription and RNA processing.Genes Dev. 2012; 26: 2119-2137Crossref PubMed Scopus (430) Google Scholar, Mayer et al., 2012Mayer A. Heidemann M. Lidschreiber M. Schreieck A. Sun M. Hintermair C. Kremmer E. Eick D. Cramer P. CTD tyrosine phosphorylation impairs termination factor recruitment to RNA polymerase II.Science. 2012; 336: 1723-1725Crossref PubMed Scopus (180) Google Scholar). At the promoter, Pol II CTD is largely unphosphorylated, but then, following initial elongation, it is converted to S5P and Y1P. This may facilitate recruitment of the capping enzyme complex. Further elongation correlates with a reduction in S5P but an increase in S2P. Transcription past the polyadenylation site then triggers recruitment of the 3′ end cleavage and polyadenylation machinery to S2P. It is possible that T4P also plays a role here, as it has recently been correlated with Pol II termination regions (Hintermair et al., 2012Hintermair C. Heidemann M. Koch F. Descostes N. Gut M. Gut I. Fenouil R. Ferrier P. Flatley A. Kremmer E. et al.Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation.EMBO J. 2012; 31: 2784-2797Crossref PubMed Scopus (108) Google Scholar, Schlackow et al., 2017Schlackow M. Nojima T. Gomes T. Dhir A. Carmo-Fonseca M. Proudfoot N.J. Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.Mol. Cell. 2017; 65: 25-38Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The kinetics of co-transcriptional splicing in budding yeast has been extensively characterized (Wallace and Beggs, 2017Wallace E.W.J. Beggs J.D. Extremely fast and incredibly close: cotranscriptional splicing in budding yeast.RNA. 2017; 23: 601-610Crossref PubMed Scopus (26) Google Scholar). The short size of yeast (S. cerevisiae) introns implies an intron definition model for the splicing mechanism (Ast, 2004Ast G. How did alternative splicing evolve?.Nat. Rev. Genet. 2004; 5: 773-782Crossref PubMed Scopus (429) Google Scholar, Barrass and Beggs, 2003Barrass J.D. Beggs J.D. Splicing goes global.Trends Genet. 2003; 19: 295-298Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, Chen and Manley, 2009Chen M. Manley J.L. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches.Nat. Rev. Mol. Cell Biol. 2009; 10: 741-754Crossref PubMed Scopus (871) Google Scholar). In particular, Pol II pausing has been observed over the 3′ ss of downstream exons in the few genes that harbor introns. This may facilitate rapid spliceosome recruitment and intron splicing (Alexander et al., 2010Alexander R.D. Innocente S.A. Barrass J.D. Beggs J.D. Splicing-dependent RNA polymerase pausing in yeast.Mol. Cell. 2010; 40: 582-593Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Furthermore, splicing in S. cerevisiae has been shown to occur within a short kinetic window following Pol II elongation through an intron and into the adjoining exon (Oesterreich et al., 2016Oesterreich F.C. Herzel L. Straube K. Hujer K. Howard J. Neugebauer K.M. Splicing of Nascent RNA Coincides with Intron Exit from RNA Polymerase II.Cell. 2016; 165: 372-381Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In mammals, the prevalence and larger size of introns instead predicts an exon definition mechanism. Binding of regulatory proteins to specific RNA sequences (splicing enhancers and inhibitors) act to select exons for splicing by promoting communication between 3′ ss and 5′ ss flanking the exon (Ast, 2004Ast G. How did alternative splicing evolve?.Nat. Rev. Genet. 2004; 5: 773-782Crossref PubMed Scopus (429) Google Scholar, Chen and Manley, 2009Chen M. Manley J.L. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches.Nat. Rev. Mol. Cell Biol. 2009; 10: 741-754Crossref PubMed Scopus (871) Google Scholar). The interplay between these regulatory factors dictates both constitutive and alternative splicing. Notably, transcription kinetics strongly impact splicing decisions. Slow Pol II elongation rates favor splicing by allowing more time for spliceosome assembly (Dujardin et al., 2014Dujardin G. Lafaille C. de la Mata M. Marasco L.E. Muñoz M.J. Le Jossic-Corcos C. Corcos L. Kornblihtt A.R. How slow RNA polymerase II elongation favors alternative exon skipping.Mol. Cell. 2014; 54: 683-690Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Muñoz et al., 2010Muñoz M.J. de la Mata M. Kornblihtt A.R. The carboxy terminal domain of RNA polymerase II and alternative splicing.Trends Biochem. Sci. 2010; 35: 497-504Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Exons in general display a higher nucleosome density, suggesting that chromatin structure acts as a kinetic barrier to Pol II elongation (Nieto Moreno et al., 2015Nieto Moreno N. Giono L.E. Cambindo Botto A.E. Muñoz M.J. Kornblihtt A.R. Chromatin, DNA structure and alternative splicing.FEBS Lett. 2015; 589: 3370-3378Crossref PubMed Scopus (30) Google Scholar, Schwartz and Ast, 2010Schwartz S. Ast G. Chromatin density and splicing destiny: on the cross-talk between chromatin structure and splicing.EMBO J. 2010; 29: 1629-1636Crossref PubMed Scopus (105) Google Scholar). We have developed a strategy for native elongation transcript sequencing using mammalian cells (mNET-seq). We found an accumulation of transcripts mapping precisely to the 3′ end of exons, as expected for intermediates formed after the first transesterification splicing reaction (Nojima et al., 2015Nojima T. Gomes T. Grosso A.R.F. Kimura H. Dye M.J. Dhir S. Carmo-Fonseca M. Proudfoot N.J. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.Cell. 2015; 161: 526-540Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). This indicates that splicing must occur within a stable complex formed between the spliceosome and Pol II. Unexpectedly, splicing intermediates were preferentially detected by antibodies specific for S5P CTD, suggesting that splicing occurs in association with this Pol II phospho-isoform. We now show biochemically and by mass spectroscopy that both protein and snRNA components of the spliceosome associate with Pol II complexes containing S5P CTD. Notably, initial cleavage of the intron creates a dominant 5′ ss intermediate that remains embedded in the Pol II-associated spliceosome. In contrast, 3′ ss intermediates corresponding to released intron lariats were detected at very low levels, suggesting fast dissociation of the spliceosome from Pol II upon completion of splicing. mNET-seq involves immunoprecipitation (IP) of human Pol II elongation complexes from chromatin solubilized by micrococcal nuclease (MNase) digestion with Pol II antibodies specific for different CTD isoforms (Nojima et al., 2015Nojima T. Gomes T. Grosso A.R.F. Kimura H. Dye M.J. Dhir S. Carmo-Fonseca M. Proudfoot N.J. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.Cell. 2015; 161: 526-540Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, Schlackow et al., 2017Schlackow M. Nojima T. Gomes T. Dhir A. Carmo-Fonseca M. Proudfoot N.J. Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.Mol. Cell. 2017; 65: 25-38Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). RNA is isolated from these complexes and sequenced by linker ligation onto the RNA 3′ ends derived either from nascent RNA in the Pol II active site or co-transcriptional RNA processing intermediates. Notably, we showed that co-transcriptional splicing is associated with Pol II phosphorylated on the CTD serine 5 position (S5P) (Nojima et al., 2015Nojima T. Gomes T. Grosso A.R.F. Kimura H. Dye M.J. Dhir S. Carmo-Fonseca M. Proudfoot N.J. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.Cell. 2015; 161: 526-540Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, Schlackow et al., 2017Schlackow M. Nojima T. Gomes T. Dhir A. Carmo-Fonseca M. Proudfoot N.J. Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.Mol. Cell. 2017; 65: 25-38Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We reasoned that, as well as sequencing IPed RNA, we should also be able to establish the protein composition of these specifically IPed Pol II complexes by mass spectroscopy (MS), termed the mNET-MS method (Figure 1A). A label-free quantitative proteomics approach (Hubner et al., 2010Hubner N.C. Bird A.W. Cox J. Splettstoesser B. Bandilla P. Poser I. Hyman A. Mann M. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions.J. Cell Biol. 2010; 189: 739-754Crossref PubMed Scopus (333) Google Scholar, Hubner and Mann, 2011Hubner N.C. Mann M. Extracting gene function from protein-protein interactions using Quantitative BAC InteraCtomics (QUBIC).Methods. 2011; 53: 453-459Crossref PubMed Scopus (84) Google Scholar) was used to determine the abundance of proteins enriched in Pol II complexes IPed with antibodies specific for S5P, S2P, and T4P CTD relative to a mock (nonspecific IgG) IP control. The fold increase of spectral intensities was compared to p values determined by t tests, as previously described (Harlen et al., 2016Harlen K.M. Trotta K.L. Smith E.E. Mosaheb M.M. Fuchs S.M. Churchman L.S. Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue.Cell Rep. 2016; 15: 2147-2158Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), generating volcano plots (Figure 1B). Significant interactors were taken to be proteins identified as enriched using a false discovery rate of 0.05. We show that complexes precipitated by all three antibodies contained Pol II subunits, transcription factors, histones, chromatin-associated proteins, RNA-binding proteins, and RNA-processing factors (Table S1). Scatterplots with fold-enrichment reveal that Pol II subunits were precipitated with similar efficiency to SUPT6H and PHRF1 (Figure 1C). Remarkably, complexes precipitated by the S5P CTD antibody contained multiple SR and SR-related proteins (SRSF7, 9,10; SCAF1, 11), as well as spliceosome components. These include Sm proteins, U1 and U2 snRNP-specific proteins, and components of spliceosomal A, B, Bact, and C complexes (Figures 1B and S1; Table S1). This was confirmed by western blot analysis of fractions IPed with S5P compared to S2P antibodies; S5P-specific enrichment was observed for the U5 snRNP protein, SNRP116, the splicing scaffold protein, PRPF8, and the U2 snRNP protein SF3B3 (Figure 1D). There are several possible reasons for this selectivity. Either spliceosomal components interact specifically with S5P CTD or, possibly, S5P and S2P epitopes display differential antibody accessibility. However, we consider this later possibility unlikely, as a recent proteomic analysis in yeast also revealed an enrichment of spliceosomal components in S5P Pol II complexes (Harlen et al., 2016Harlen K.M. Trotta K.L. Smith E.E. Mosaheb M.M. Fuchs S.M. Churchman L.S. Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue.Cell Rep. 2016; 15: 2147-2158Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). As an alternative approach to studying the interaction between Pol II and the spliceosome, chromatin was solubilized by MNase treatment and then fractionated on a 10%–30% glycerol gradient followed by protein extraction from 22 separated fractions (Figure 1E). These were subjected to western blotting with antibodies against total, S2P, and S5P Pol II as well as U5 snRNP protein SNRP116 and histone H3 (Figure 1F). Significant amounts of S2P, S5P Pol II, and histone H3 were detected in the middle and heavy fractions. However, the heavy fraction contained most of the snRNP116 (Figure 1F). Further analysis with a wider range of antibodies against pooled light, middle, and heavy fractions confirms that U5 snRNP proteins were predominantly associated with the heavy fraction (Figure 1G). In contrast, U2 snRNP proteins appeared similarly distributed in the middle and heavy fractions, and U1 snRNP proteins were predominantly associated with the middle fraction (Figure 1G). The histone mark H3K9me2, which is typically associated with transcriptional repression, was predominantly in the middle fraction, whereas the histone elongation mark H3K36me3 was in both the middle and heavy fractions. These results indicate that the heavy fraction contains spliceosome-associated S5P Pol II-elongating polymerases on a chromatin template. Since S5P CTD modified Pol II interacts with protein components of the spliceosome, we also analyzed spliceosomal snRNA. We reasoned that mNET-seq should detect the free 3′ OH ends of mature spliceosomal snRNA associated with Pol II. Indeed, an accumulation of mNET-seq signal was found mapping to the 3′ ends of U1, U2, U4, and U5 snRNAs (Figure 2A). As expected, no 3′ end peak was detected in U6 snRNA, which contains a 2′,3′ cyclic phosphate terminal group at the 3′ end (Lund and Dahlberg, 1992Lund E. Dahlberg J.E. Cyclic 2′, 3′ -phosphates and nontemplated nucleotides at the 3′ end of spliceosomal U6 small nuclear RNA’s.Science. 1992; 255: 327-330Crossref PubMed Scopus (110) Google Scholar). Lower mNET-seq peaks were also found at the 3′ ends of the minor spliceosome U11, U12, and U4atac snRNAs, but not in U6atac snRNA (Figure 2B). A weak 3′ end signal was further detected for U7 snRNA, which is associated with histone pre-mRNA 3′ end processing. As expected, no 3′ end peak was detected for U3 snRNA, which is involved in the processing of Pol I-synthesized pre-rRNA (Figures 2C and S2A). To confirm the specificity of these results, we tested whether the spliceosome snRNAs’ 3′ end signal was sensitive to treatment with the strong detergent empigen. We have previously employed empigen to separate RNA processing complexes from Pol II (Schlackow et al., 2017Schlackow M. Nojima T. Gomes T. Dhir A. Carmo-Fonseca M. Proudfoot N.J. Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.Mol. Cell. 2017; 65: 25-38Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Notably mNET-seq signals of both major and minor spliceosome snRNAs 3′ ends were substantially diminished (Figure 2D). A more detailed comparison of the association of mature spliceosomal snRNA (based on mNET-seq 3′ end reads) for all phospho CTD isoforms of Pol II revealed higher signal with S5P CTD antibody for U2, U5, and the minor spliceosome snRNA. In contrast, U1 snRNA showed similar read levels for all five phospho CTD isoforms (Figure 2E). To complete this mNET-seq analysis of spliceosome-associated RNA, we also tested the distribution of the 5′ ss splicing intermediate. We first confirmed that RNA fragments mapping to the 5′ ss are predominantly detected by S5P CTD antibody as opposed to S2P, Y1P, T4P and S7P antibodies (Figure S2B). We also compared mNET-seq profiles with different CTD S5P-specific antibodies; MABI0603 (MBL international), routinely used in our mNET-seq/S5P analysis, and ab5131 (Abcam), often used in Pol II ChIP analysis. Similar profiles of 5′ ss peaks were obtained (Figures S2C and S2D), consistent with our previous ChIP analysis that showed their same specificity for S5P CTD (Nojima et al., 2015Nojima T. Gomes T. Grosso A.R.F. Kimura H. Dye M.J. Dhir S. Carmo-Fonseca M. Proudfoot N.J. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.Cell. 2015; 161: 526-540Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). We next generated mNET-seq/S5P and S2P libraries from the heavy and middle fractions of the glycerol gradient of MNase-digested chromatin. Notably, the 5′ ss splicing intermediate was only detected from the heavy S5P library as shown for TARS (Figure S2E) and in a metagene profile of 40,896 aligned exon intron junctions corresponding to spliced events (Figure S2F). These results confirm the existence of a complex between Pol II and the spliceosome in heavy fractions. Finally, since the 5′ ss splicing intermediate detected by mNETseq/S5P is empigen sensitive (Figure S2G), we predict that the 5′ ss RNA is associated with the spliceosome rather than with the Pol II active site. In summary, these data strongly argue that phosphorylation of CTD serine 5 residues is not restricted to transcription initiation but is also present during elongation and co-transcriptional splicing. It is clear from the range of data presented (Figures 1, 2, S1, and S2) that catalytically active spliceosome stably associates with S5P CTD Pol II. We next employed mNET-seq to investigate the extent of nascent transcript interaction with both Pol II and the spliceosome. Since this procedure involves MNase digestion before RNA purification and library preparation, it can be used for Pol II and spliceosome footprinting analysis akin to ribosome footprinting. Previously, we size selected RNA of 30–100 nt for library preparation (Nojima et al., 2015Nojima T. Gomes T. Grosso A.R.F. Kimura H. Dye M.J. Dhir S. Carmo-Fonseca M. Proudfoot N.J. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing.Cell. 2015; 161: 526-540Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, Schlackow et al., 2017Schlackow M. Nojima T. Gomes T. Dhir A. Carmo-Fonseca M. Proudfoot N.J. Distinctive Patterns of Transcription and RNA Processing for Human lincRNAs.Mol. Cell. 2017; 65: 25-38Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To explore a larger RNA size range, we prepared new mNETseq/S5P libraries using long-sized (60–160 nt) or short-sized (20–60 nt) RNA fractions with a read length of 150 bp and 75 bp, respectively (Figure 3A). To analyze Pol II footprints independently of spliceosome assembly, we examined the length of reads mapping to a region from the TSS to +150 nucleotides in first exons that were at least 200 nt long. Most reads (> 85%) obtained from the short-sized RNA fractions were 18–40 nt long with a peak at 27 nt, whereas those for the long-sized RNA fractions were 40–100 nt long with a peak at 60 nt (Figure 3B). We infer that Pol II complexes protect a total of either 27 or 60 nascent transcript nucleotides. Previous structural data indicate that nascent RNA must be at least 15 nt in length to reach the Pol II surface (Andrecka et al., 2009Andrecka J. Treutlein B. Arcusa M.A. Muschielok A. Lewis R. Cheung A.C. Cramer P. Michaelis J. Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex.Nucleic Acids Res. 2009; 37: 5803-5809Crossref PubMed Scopus (77) Google Scholar, Kettenberger et al., 2004Kettenberger H. Armache K.J. Cramer P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS.Mol. Cell. 2004; 16: 955-965Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar) and 17–23 nt to reach the active site of the capping enzyme, which binds Pol II at the end of the RNA exit tunnel (Martinez-Rucobo et al., 2015Martinez-Rucobo F.W. Kohler R. van de Waterbeemd M. Heck A.J. Hemann M. Herzog F. Stark H. Cramer P. Molecular Basis of Transcription-Coupled Pre-mRNA Capping.Mol. Cell. 2015; 58: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). To determine whether binding of the capping enzyme to Pol II explains the 20–30 nt footprint, we compared the length of reads mapping to the same exons on the region from the TSS to +50 nts and from +100 to +150 nts (Figure S3A). A similar footprint was obtained irrespective of read position with mNET-seq libraries prepared after IP with either S5P and S2P antibodies (Figure S3A). This suggests that once nascent transcripts emerge from the polymerase exit channel, they are protected from MNase by RNA-binding proteins such as the capping enzyme. The larger 60-nt footprint obtained from long-sized RNA fractions (Figure 3B) was further characterized. Analysis of mNET-seq profiles obtained from the long-sized RNA fractions no longer shows the characteristic peaks corresponding to promoter proximal transcriptional pausing that typically occurs within the first 50 bp of the transcription start site (TSS). This is shown both for the TSS region of DAP3 (Figure 3C) and a metagene profile of long and short mNET-seq/S5P TSS reads (Figure 3D). We conclude that the mNET-seq protocol distinguishes two types of elongating Pol II complexes, a smaller one corresponding to a nascent RNA footprint of 27 nt and a larger one protecting approximately 60 nt of nascent transcripts (Figure 3E). Next, we examined the read length of splicing intermediates in mNETseq/S5P and S2P libraries prepared from the short and long-sized RNA fractions (Figure S3B). The results show that the most frequent length of both 5′ ss intermediates (i.e., exons that have been cleaved at the 5′ ss but not yet ligated) and 3′ ss intermediates (i.e., released intron lariats) is 30–32 nt in the short libraries and 57–60 nt in the long libraries. As splicing intermediates are spliceosome-bound, we conclude that these fragments result from the footprinting of splicing complexes associated with S5P and S2P CTD Pol II. Notably, a very recent analysis of RNA co-purifying with late-stage spliceosomes similarly revealed that splicing complexes protect a total of approximately 56 nucleotides (Chen et al., 2018Chen W. Moore J. Ozadam H. Shulha H.P. Rhind N. Weng Z. Moore M.J. Transcriptome-wide Interrogation of the Functional Intronome by Spliceosome Profiling.Cell. 2018; 173: 1031-1044.e1013Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). We show above (Figures 1 and 2) that the spliceosome forms a complex with Pol II. We therefore tested if splicing can occur immediately after the 3′ ss in a nascent transcript emerges at the Pol II surface. We reasoned that, if this was the case, then mNET-seq should detect spliced nascent transcripts. Analysis of mNET-seq libraries prepared from the short RNA fraction revealed spliced exon reads corresponding to approximately 300 splicing events, while over 20,000 splicing events were detected in libraries prepared from the long RNA fraction (Figure 3F). Most spliced events detected by spliced exon reads were predominantly observed in mNET-seq/S5P libraries (Figure 3G), consistent with our data indicating that complexes of Pol II and associated catalytically active spliceosomes are preferentially IPed by S5P CTD antibodies (Figures 1, 2, S1, and S2). At this stage in our analysis, we added additional mNET-seq/S5P libraries for the mouse lymphoid cell line TAP to generalize our results on co-transcriptional splicing to a different mammal and cell type. We also performed heat-map analysis of mNET-seq signals and detected more S5P signals on spliced exons than other phospho CTD isoforms (Figures S3C–S3E). Notably these mNET-seq/S5P signals are resistant to empigen, suggesting that they are derived from nascent transcripts connected to the Pol II active site (Figure S3F). These empigen-resistant mNET-seq/S5P signals do not show any bias across exons (Figure S3G). This indicates that S5P CTD Pol II pausing is exon position independent. In contrast, the vast majority of the spliced reads in human and murine libraries display empigen sensitivity (Figure 3H), showing that they are nascent RNAs associated with Pol II (presumably within the spliceosome) but not from the Pol II active site. The combined analysis of human and murine libraries led us to appreciate that cDNA primers used in next-generation sequencing can internally prime on spliceosome-derived spliced transcripts as well as priming on the linker ligated onto RNA 3′ ends (either 5′ ss intermediates or Pol II active site RNA). This was evident from a conserved 4-nt sequence (TGGA from Illu" @default.
• W2897066913 created "2018-10-26" @default.
• W2897066913 creator A5004220955 @default.
• W2897066913 creator A5005381817 @default.
• W2897066913 creator A5005976170 @default.
• W2897066913 creator A5013646145 @default.
• W2897066913 creator A5047722486 @default.
• W2897066913 creator A5083230780 @default.
• W2897066913 date "2018-10-01" @default.
• W2897066913 modified "2023-10-14" @default.
• W2897066913 title "RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing" @default.
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