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The regulation and function of post-transcriptional RNA splicing

Nature Reviews Genetics volume 26, pages 378–394 (2025)Cite this article

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Abstract

Eukaryotic RNA transcripts undergo extensive processing before becoming functional messenger RNAs, with splicing being a critical and highly regulated step that occurs both co-transcriptionally and post-transcriptionally. Recent analyses have revealed, with unprecedented spatial and temporal resolution, that up to 40% of mammalian introns are retained after transcription termination and are subsequently removed largely while transcripts remain chromatin-associated. Post-transcriptional splicing has emerged as a key layer of gene expression regulation during development, stress response and disease progression. The control of post-transcriptional splicing regulates protein production through delayed splicing and nuclear export, or nuclear retention and degradation of specific transcript isoforms. Here, we review current methodologies for detecting post-transcriptional splicing, discuss the mechanisms controlling the timing of splicing and examine how this temporal regulation affects gene expression programmes in healthy cells and in disease states.

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Fig. 1: Overview of co-transcriptional and post-transcriptional splicing.
Fig. 2: Experimental approaches to study post-transcriptional splicing.
Fig. 3: Splicing as a rate-limiting step for chromatin release.
Fig. 4: Types of post-transcriptional splicing and connection to transcript fate.
Fig. 5: Regulation of post-transcriptional splicing.

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Acknowledgements

The authors thank H. Merens, A.-M. Raicu and L. Hansen for critical reading of the manuscript. This work was supported by a Parkinson’s Foundation Postdoctoral fellowship to I.L.P.

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  1. These authors contributed equally: Karine Choquet, Ines L. Patop.

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  1. Department of Biochemistry and Functional Genomics, University of Sherbrooke, Sherbrooke, Quebec, Canada

    Karine Choquet

  2. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Ines L. Patop & L. Stirling Churchman

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  1. Karine Choquet
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Glossary

Alternative splicing

A regulated process by which different combinations of exons and/or introns from a single gene can be included or excluded in the final mRNA, allowing multiple protein isoforms to be produced from a single gene.

Alternative untranslated regions

(UTRs). Different versions of untranslated regions that can be included in the final mRNA through alternative splicing, alternative polyadenylation or alternative transcription start sites.

Branch point

An intronic adenosine, typically located 18–40 nt upstream of the 3′ splice site, whose 2′-OH group attacks the 5′ splice site in the first step of splicing, creating a characteristic branched intermediate (lariat).

Cleavage and polyadenylation

(CPA). The process of cutting the primary transcript at a specific site and adding a poly(A) tail to create the 3′-end of mature mRNA.

Cleavage and polyadenylation sites

(PAS). Specific sequences in the pre-mRNA that signal where the transcript should be cleaved and polyadenylated.

Detained intron

A class of introns that are retained in nucleus-localized transcripts until specific signals trigger their removal and export of the transcript from the nucleus, serving as a regulatory mechanism for gene expression.

m6A

N6-methyladenosine, an RNA chemical modification consisting of a methyl group added to the nitrogen at position 6 of adenosine.

Metabolic labelling

A technique in which cells are grown with nucleotide analogues that are incorporated into newly synthesized RNA, which can be specifically isolated or detected using biochemical methods, allowing to track new RNA synthesis and processing.

MicroRNAs

Small non-coding RNAs (21–23 nt) that regulate gene expression post-transcriptionally, generally through binding sites in 3′ untranslated regions.

mRNA

A class of RNA molecules that unlike non-coding RNAs carry coding information and are translated into proteins by ribosomes.

Nonsense-mediated decay

(NMD). A quality control mechanism that degrades mRNAs containing premature stop codons in the cytoplasm.

Nuclear degradation

The breakdown of RNA molecules within the nucleus, serving as a quality control mechanism and regulatory process. This occurs through multiple pathways including the nuclear exosome.

Nuclear speckles

Membrane-less nuclear compartments enriched in pre-mRNA splicing factors, RNA-processing factors and partially processed mRNAs.

PUND

Genes encoding transcripts that are predicted to undergo nuclear degradation rather than be processed into mRNA based on subcellular fractionation, metabolic labelling, mathematical modelling and/or knockdown of nuclear exosome subunits.

RNA polymerase II

(Pol II). The enzyme responsible for transcribing all eukaryotic protein-coding genes and many non-coding RNA genes. Its largest subunit contains a C-terminal domain that undergoes dynamic phosphorylation during transcription, coordinating various RNA processing events.

RNA-binding proteins

(RBPs). Proteins that bind to RNA, typically by recognizing specific sequences or structures, and regulate processes including splicing, polyadenylation, export, localization, stability and translation.

Serine–arginine (SR) proteins

A family of proteins that have crucial roles in constitutive and alternative splicing. They are characterized by one or more RNA recognition motif(s) and a domain rich in serine–arginine dipeptides (RS domain). Dynamic phosphorylation of the RS domain regulates their activity, localization and function.

Splice sites

Specific sequences at the start (5′-end) and end (3′-end) of introns that are recognized by the spliceosome.

Subcellular fractionation

Separation of subcellular compartments through differential centrifugation and/or biochemical extraction, yielding cytoplasmic, nucleoplasmic and chromatin-associated fractions.

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Choquet, K., Patop, I.L. & Churchman, L.S. The regulation and function of post-transcriptional RNA splicing. Nat Rev Genet 26, 378–394 (2025). https://doi.org/10.1038/s41576-025-00836-z

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