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POINT technology illuminates the processing of polymerase-associated intact nascent transcripts

ABSTRACT: Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE159326 | GEO | 2021/03/19

REPOSITORIES: GEO

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Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing

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2019-12-12 | GSE123191 | GEO

Targeted protein degradation reveals Pol II heterogeneity and functional diversity

Project description:RNA polymerase II (Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription process and preferentially regulate RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to the depletion of different Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of Pol II and different genes appear to depend on some of the subunits.

2022-08-09 | PXD035866 | iProX

Alternative RNA Stuctures Formed During Transcription Depend on Elongation Rate and Modify RNA processing

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2021-02-09 | GSE149018 | GEO

Identification of CDK9 targets using DRB and quantitative phosphoproteomics

Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allow the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.

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Revealing nascent RNA processing dynamics with nano-COP

Project description:During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3’-end cleavage, and polyadenylation. Here, we describe nanopore analysis of CO-transcriptional Processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing (NET-seq), which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3’ ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read–based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 days.

2021-01-06 | GSE154079 | GEO

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation (expression)

Project description:The rate of RNA polymerase II (pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing is not well understood. We performed experiments to perturb pol II elongation and then globally compared alternative splicing patterns with genome-wide pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with pol II elongation inhibition-dependent changes in alternative splicing. Under conditions that interfere with pol II elongation, including cell stress, increased pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, alternative splicing and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. In order to identify alternative splicing events influenced by changes in pol II elongation, we performed quantitative alternative splicing microarray profiling (Pan et al., 2004 (PMID 15610736); Shai et al., 2006 (PMID 16403798)) of RNA isolated from stimulated Jurkat T lymphoma cells, cultured separately in the presence or absence of two different drugs that can inhibit pol II elongation: 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) and camptothecin.

2010-12-09 | E-GEOD-25287 | biostudies-arrayexpress

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation (ChIP-Seq)

Project description:The rate of RNA polymerase II (pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing is not well understood. We performed experiments to perturb pol II elongation and then globally compared alternative splicing patterns with genome-wide pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with pol II elongation inhibition-dependent changes in alternative splicing. Under conditions that interfere with pol II elongation, including cell stress, increased pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, alternative splicing and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels. To monitor pol II distributions, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed using an anti-pol II antibody (4H8) and cross-linked chromatin preparations from Jurkat cells, treated with or without pol II elongation inhibitor 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) at 10 and 25 ug/ml respectively prior to phorbol 12-myristate 13-acetate (PMA) stimulation, for 5000+ alternative splicing events.

2010-12-09 | E-GEOD-25494 | biostudies-arrayexpress

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation (ChIP-Seq)

2010-12-09 | GSE25494 | GEO

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation (expression)

2010-12-09 | GSE25287 | GEO

Widespread transcriptional readthrough caused by Nab2 depletion leads to chimeric transcripts with retained introns

Project description:Nascent RNA sequencing has recently revealed that pre-mRNA splicing can occur shortly after the intron emerges from RNA polymerase II (Pol II). Differences in co-transcriptional splicing profiles suggest regulation by cis- and/or trans-acting factors. Here we used Single Molecule Intron Tracking (SMIT) in budding yeast to identify a cohort of regulators by machine learning. One candidate, Nab2, displayed reduced co-transcriptional splicing of some pre-mRNAs when depleted. Unexpectedly, these splicing defects were attributable to readthrough transcription, which was revealed by long read sequencing of nascent RNA; individual readthrough transcripts induced by Nab2 depletion sometimes spanned multiple genes. Thus, Nab2 regulation of splicing was indirect. Moreover, unspliced transcripts displayed downstream readthrough in both control and Nab2-depleted cells, highlighting the coupling between splicing and 3′ end formation. We conclude that Nab2 is required for proper 3′ end processing, which ensures both transcription termination as well as proper splicing of downstream genes.

2020-10-27 | GSE156133 | GEO

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