Pre-mRNA Splicing in the Nuclear Landscape

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Abstract

Eukaryotic gene expression requires the cumulative activity of multiple molecular machines to synthesize and process newly transcribed pre-messenger RNA. Introns, the noncoding regions in pre-mRNA, must be removed by the spliceosome, which assembles on the pre-mRNA as it is transcribed by RNA polymerase II (Pol II). The assembly and activity of the spliceosome can be modulated by features including the speed of transcription elongation, chromatin, post-translational modifications of Pol II and histone tails, and other RNA processing events like 5′-end capping. Here, we review recent work that has revealed cooperation and coordination among co-transcriptional processing events and speculate on new avenues of research. We anticipate new mechanistic insights capable of unraveling the relative contribution of coupled processing to gene expression.

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Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing

Takayuki Nojima, Tomás Gomes, Ana Rita Fialho Grosso … (2015)

Summary Transcription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5′splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

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RNA Splicing by the Spliceosome

Max Wilkinson, Clément Charenton, Kiyoshi Nagai (2020)

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5′ splice site (5′SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5′SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5′SS, producing a free 5′ exon. Removal of the BP adenosine from the active site allows the 3′SS to bind, so that the 5′ exon attacks the 3′SS to produce mature mRNA and an excised lariat intron. Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs

Hagen Tilgner, David G. Knowles, Rory Johnson … (2012)

Splicing remains an incompletely understood process. Recent findings suggest that chromatin structure participates in its regulation. Here, we analyze the RNA from subcellular fractions obtained through RNA-seq in the cell line K562. We show that in the human genome, splicing occurs predominantly during transcription. We introduce the coSI measure, based on RNA-seq reads mapping to exon junctions and borders, to assess the degree of splicing completion around internal exons. We show that, as expected, splicing is almost fully completed in cytosolic polyA+ RNA. In chromatin-associated RNA (which includes the RNA that is being transcribed), for 5.6% of exons, the removal of the surrounding introns is fully completed, compared with 0.3% of exons for which no intron-removal has occurred. The remaining exons exist as a mixture of spliced and fewer unspliced molecules, with a median coSI of 0.75. Thus, most RNAs undergo splicing while being transcribed: “co-transcriptional splicing.” Consistent with co-transcriptional spliceosome assembly and splicing, we have found significant enrichment of spliceosomal snRNAs in chromatin-associated RNA compared with other cellular RNA fractions and other nonspliceosomal snRNAs. CoSI scores decrease along the gene, pointing to a “first transcribed, first spliced” rule, yet more downstream exons carry other characteristics, favoring rapid, co-transcriptional intron removal. Exons with low coSI values, that is, in the process of being spliced, are enriched with chromatin marks, consistent with a role for chromatin in splicing during transcription. For alternative exons and long noncoding RNAs, splicing tends to occur later, and the latter might remain unspliced in some cases.

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Journal

Journal ID (nlm-journal-id): 1256107

Journal ID (pubmed-jr-id): 3100

Journal ID (nlm-ta): Cold Spring Harb Symp Quant Biol

Journal ID (iso-abbrev): Cold Spring Harb. Symp. Quant. Biol.

Title: Cold Spring Harbor symposia on quantitative biology

ISSN (Print): 0091-7451

ISSN (Electronic): 1943-4456

Publication date Nihms-submitted: 18 June 2020

Publication date (Electronic): 03 June 2020

Publication date (Print): 2019

Publication date PMC-release: 28 July 2020

Volume: 84

Pages: 11-20

Affiliations

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA

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Correspondence: karla.neugebauer@ 123456yale.edu

Article

Manuscript ID: NIHMS1604922

DOI: 10.1101/sqb.2019.84.040402

PMC ID: 7384967

PubMed ID: 32493763

SO-VID: b3bb3ce2-6c30-48a0-9a1c-a91ebcee7d49

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This article is distributed under the terms of the Creative Commons Attribution-NonCommercial License, which permits reuse and redistribution, except for commercial purposes, provided that the original author and source are credited.

Pre-mRNA Splicing in the Nuclear Landscape

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Most cited references 105

Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing

RNA Splicing by the Spliceosome

Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs

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