Recurrent Spliceosome Mutations in Cancer: Mechanisms and Consequences of Aberrant Splice Site Selection

Splicing alterations have been widely documented in tumors where the proliferation and dissemination of cancer cells is supported by the expression of aberrant isoform variants. Splicing is catalyzed by the spliceosome, a ribonucleoprotein complex that orchestrates the complex process of intron removal and exon ligation. In recent years, recurrent hotspot mutations in the spliceosome components U1 snRNA, SF3B1, and U2AF1 have been identified across different tumor types. Such mutations in principle are highly detrimental for cells as all three spliceosome components are crucial for accurate splice site selection: the U1 snRNA is essential for 3′ splice site recognition, and SF3B1 and U2AF1 are important for 5′ splice site selection. Nonetheless, they appear to be selected to promote specific types of cancers. Here, we review the current molecular understanding of these mutations in cancer, focusing on how they influence splice site selection and impact on cancer development.

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Post-catalytic spliceosome structure reveals mechanism of 3'-splice site selection

10.1101/212811 ◽

2017 ◽

Author(s):

Max E. Wilkinson ◽

Sebastian M. Fica ◽

Wojciech P. Galej ◽

Christine M. Norman ◽

Andrew J. Newman ◽

...

Keyword(s):

Branch Point ◽

Splice Site ◽

Site Selection ◽

Catalytic Mechanism ◽

Splice Site Selection ◽

Terminal Domain ◽

Exon Ligation ◽

Site Recognition ◽

U5 Snrna ◽

Insight Into

AbstractIntrons are removed from eukaryotic mRNA precursors by the spliceosome in two transesterification reactions – branching and exon ligation. Following branching, the 5'-exon remains paired to U5 snRNA loop 1, but the mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7Å cryo-EM structure of the yeast P complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognised through non-Watson-Crick pairing with the 5'-splice site and the branch point adenosine. A conserved loop of Prp18 together with the α-finger and the RNaseH domain of Prp8 clamp the docked 3'-splice site and 3'-exon. The step 2 factors Prp18 and Slu7 and the C-terminal domain of Yju2 stabilise a conformation competent for 3'-splice site docking and exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides of the introns and provides insight into the catalytic mechanism of exon ligation.

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Exon mutations uncouple 5′ splice site selection from U1 snRNA pairing

Cell ◽

10.1016/0092-8674(90)90457-p ◽

1990 ◽

Vol 63 (3) ◽

pp. 619-629 ◽

Cited By ~ 25

Author(s):

Bertrand Séraphin ◽

Michael Rosbash

Keyword(s):

Splice Site ◽

Site Selection ◽

Splice Site Selection ◽

U1 Snrna

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Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.01071-09 ◽

2010 ◽

Vol 30 (8) ◽

pp. 1878-1886 ◽

Cited By ~ 24

Author(s):

Martin J. Hicks ◽

William F. Mueller ◽

Peter J. Shepard ◽

Klemens J. Hertel

Keyword(s):

Splice Site ◽

Site Selection ◽

Regulatory Element ◽

Regulatory Elements ◽

Splice Sites ◽

Base Pairing ◽

Splice Site Selection ◽

U1 Snrna ◽

Pairing Potential

ABSTRACT Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

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The exon junction complex and intron removal prevent re-splicing of mRNA

PLoS Genetics ◽

10.1371/journal.pgen.1009563 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009563

Author(s):

Brian Joseph ◽

Eric C. Lai

Keyword(s):

Gene Expression ◽

Splice Site ◽

Site Selection ◽

Exon Junction Complex ◽

Splice Sites ◽

Splice Site Selection ◽

Exon Junction ◽

Intron Removal ◽

Cryptic Splice Sites

Accurate splice site selection is critical for fruitful gene expression. Recently, the mammalian EJC was shown to repress competing, cryptic, splice sites (SS). However, the evolutionary generality of this remains unclear. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic SS, even though most bear weak splicing motifs and are seemingly incompetent. Mechanistically, the EJC directly conceals cryptic splicing elements by virtue of its position-specific recruitment, preventing aberrant SS definition. Unexpectedly, we discover the EJC inhibits scores of regenerated 5’ and 3’ recursive SS on segments that have already undergone splicing, and that loss of EJC regulation triggers faulty resplicing of mRNA. An important corollary is that certain intronless cDNA constructs yield unanticipated, truncated transcripts generated by resplicing. We conclude the EJC has conserved roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

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The Exon Junction Complex and intron removal prevents resplicing of mRNA

10.1101/2020.07.31.231498 ◽

2020 ◽

Author(s):

Brian Joseph ◽

Eric C. Lai

Keyword(s):

Gene Expression ◽

Splice Site ◽

Site Selection ◽

Exon Junction Complex ◽

Splice Sites ◽

Splice Site Selection ◽

Exon Junction ◽

Exon Junctions ◽

Intron Removal ◽

Cryptic Splice Sites

AbstractAccurate splice site selection is critical for fruitful gene expression. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic splice sites (SS), even though majority of these bear weak splicing motifs and appear incompetent. Mechanistically, the EJC directly conceals splicing elements through position-specific recruitment, preventing SS definition. We note that intron removal using strong, canonical SS yields AG|GU signatures at exon-exon junctions. Unexpectedly, we discover that scores of these minimal exon junction sequences are in fact EJC-suppressed 5’ and 3’ recursive SS, and that loss of EJC regulation from such transcripts triggers faulty mRNA resplicing. An important corollary is that intronless cDNA expression constructs from aforementioned targets yield high levels of unanticipated, truncated transcripts generated by resplicing. Consequently, we conclude the EJC has ancestral roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

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Suppressor U1 snRNAs in Drosophila.

Genetics ◽

10.1093/genetics/138.2.365 ◽

1994 ◽

Vol 138 (2) ◽

pp. 365-378 ◽

Cited By ~ 1

Author(s):

P C Lo ◽

D Roy ◽

S M Mount

Keyword(s):

Splice Site ◽

Splice Site Mutation ◽

Base Pairing ◽

Site Mutation ◽

Splice Site Selection ◽

Transfected Cells ◽

U1 Snrna ◽

Base Pairing Interaction ◽

Multicellular Organisms ◽

Site Recognition

Abstract Although the role of U1 small nuclear RNAs (snRNAs) in 5' splice site recognition is well established, suppressor U1 snRNAs active in intact multicellular animals have been lacking. Here we describe suppression of a 5' splice site mutation in the Drosophila melanogaster white gene (wDR18) by compensatory changes in U1 snRNA. Mutation of positions -1 and +6 of the 5' splice site of the second intron (ACG[GTGAGT to ACC]GTGAGC) results in the accumulation of RNA retaining this 74-nucleotide intron in both transfected cells and transgenic flies. U1-3G, a suppressor U1 snRNA which restores base-pairing at position +6 of the mutant intron, increases the ratio of spliced to unspliced wDR18 RNA up to fivefold in transfected Schneider cells and increases eye pigmentation in wDR18 flies. U1-9G, which targets position -1, suppresses wDR18 in transfected cells less well. U1-3G,9G has the same effect as U1-3G although it accumulates to lower levels. Suppression of wDR18 has revealed that the U1b embryonic variant (G134 to U) is active in Schneider cells and pupal eye discs. However, the combination of 9G with 134U leads to reduced accumulation of both U1b-9G and U1b-3G,9G, possibly because nucleotides 9 and 134 both participate in a potential long-range intramolecular base-pairing interaction. High levels of functional U1-3G suppressor reduce both viability and fertility in transformed flies. These results show that, despite the difficulties inherent in stably altering splice site selection in multicellular organisms, it is possible to obtain suppressor U1 snRNAs in flies.

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Faculty Opinions recommendation of Stop codons affect 5' splice site selection by surveillance of splicing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1006506.91656 ◽

2002 ◽

Author(s):

Miles Wilkinson

Keyword(s):

Splice Site ◽

Site Selection ◽

Splice Site Selection ◽

Stop Codons

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The anti-angiogenic isoforms of VEGF in health and disease

Biochemical Society Transactions ◽

10.1042/bst0371207 ◽

2009 ◽

Vol 37 (6) ◽

pp. 1207-1213 ◽

Cited By ~ 76

Author(s):

Yan Qiu ◽

Coralie Hoareau-Aveilla ◽

Sebastian Oltean ◽

Steven J. Harper ◽

David O. Bates

Keyword(s):

Splice Site ◽

Site Selection ◽

Age Related Macular Degeneration ◽

Splicing Factor ◽

Splice Site Selection ◽

Age Related ◽

Normal Human ◽

Radical Revision

Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys–Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.

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