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CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing [RNA-Seq]

ABSTRACT: Transcription and co-transcriptional pre-mRNA splicing are kinetically coupled as shown by the widespread effects of transcription speed on alternative splicing. The molecular basis for kinetic coupling is incompletely understood, but one potential mechanism is through elongation rate dependent alternative folding pathways of the nascent pre-mRNA. To search for RNA structures in nascent pre-mRNA, we modified SPLASH (1) (Sequencing of Psoralen Crosslinked, Ligated And Selected Hybrids) for use with Chromatin Associated RNA. We applied this new method called CAR-SPLASH to cells expressing WT and slow mutant pol II and identified >3000 intragenic RNA structures of which >400 are proximal to splice sites. ASO disruption of several structures identified by CAR-SPLASH that sequester splice sites changed alternative splicing outcomes in vivo. We identified three examples of novel regulatory elements we designate “RNA kinetic switches”. ASO disruption of these structures modified alternative splicing of NISCH Exon 18 GAK Exon 7 and MEGF8 Exon 24 in a way that depended on the rate of transcription elongation. These results demonstrate that individual RNA structures can mediate kinetic coupling between transcription and pre-mRNA splicing and that nascent RNA structures can serve as targets for splice modifying ASO’s.

ORGANISM(S): Homo sapiens

PROVIDER: GSE288524 | GEO | 2025/09/14

REPOSITORIES: GEO

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CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing [smallRNA-Seq]

Project description:Transcription and co-transcriptional pre-mRNA splicing are kinetically coupled as shown by the widespread effects of transcription speed on alternative splicing. The molecular basis for kinetic coupling is incompletely understood, but one potential mechanism is through elongation rate dependent alternative folding pathways of the nascent pre-mRNA. To search for RNA structures in nascent pre-mRNA, we modified SPLASH (1) (Sequencing of Psoralen Crosslinked, Ligated And Selected Hybrids) for use with Chromatin Associated RNA. We applied this new method called CAR-SPLASH to cells expressing WT and slow mutant pol II and identified >3000 intragenic RNA structures of which >400 are proximal to splice sites. ASO disruption of several structures identified by CAR-SPLASH that sequester splice sites changed alternative splicing outcomes in vivo. We identified three examples of novel regulatory elements we designate “RNA kinetic switches”. ASO disruption of these structures modified alternative splicing of NISCH Exon 18 GAK Exon 7 and MEGF8 Exon 24 in a way that depended on the rate of transcription elongation. These results demonstrate that individual RNA structures can mediate kinetic coupling between transcription and pre-mRNA splicing and that nascent RNA structures can serve as targets for splice modifying ASO’s.

2025-09-14 | GSE288523 | GEO

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Project description:Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing.

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CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing [RNA-Seq]

Project description:CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing [RNA-Seq]

| PRJNA1217922 | ENA

CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing [smallRNA-Seq]

Project description:CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing [smallRNA-Seq]

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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.

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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.

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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)

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Heritability of alternative splicing in the human genome

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