Precursor RNA Structural Patterns at SF3B1 Mutation Sensitive Cryptic 3' Splice Sites

Overview

Journal bioRxiv

Date 2025 Mar 3

PMID 40027643

Authors

Austin Herbert

Abigail Hatfield

Alexandra Randazza

Valeria Miyamoto

Katie Palmer

Lela Lackey

Affiliations

Soon will be listed here.

Abstract

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3' splice site selection. SF3B1 mutation is common in myelodysplastic syndrome and other blood disorders. The most common mutation in SF3B1 is K700E, a lysine to glutamic acid change within the pre-mRNA interacting heat repeat domain. A hallmark of SF3B1 mutation is an increased use of cryptic 3' splice sites; however, the properties distinguishing SF3B1-sensitive splice junctions from other alternatively spliced junctions are unknown. We identify a subset of 192 core splice junctions that are mis-spliced with SF3B1 K700E mutation. We use our core set to test whether SF3B1-sensitive splice sites are different from control cryptic 3' splice sites via RNA structural accessibility. As a comparison, we define a set of SF3B1-resistant splice junctions with cryptic splice site use that does not change with SF3B1 K700E mutation. We find sequence differences between SF3B1-sensitive and SF3B1-resistant junctions, particularly at the cryptic sites. SF3B1-sensitive cryptic 3' splice sites are within an extended polypyrimidine tract and have lower splice site strength scores. We develop experimental RNA structure data for 83 SF3B1-sensitive junctions and 39 SF3B1-resistant junctions. We find that the pattern of structural accessibility at the NAG splicing motif in cryptic and canonical 3' splice sites is similar. In addition, this pattern can be found in both SF3B1-resistant and SF3B1-sensitive junctions. However, SF3B1-sensitive junctions have cryptic splice sites that are less structurally distinct from the canonical splice sites. In addition, SF3B1-sensitive splice junctions are overall more flexible than SF3B1-resistant junctions. Our results suggest that the SF3B1-sensitive splice junctions have unique structure and sequence properties, containing poorly differentiated, weak splice sites that lead to altered 3' splice site recognition in the presence of SF3B1 mutation.

References

Golomb L, Bublik D, Wilder S, Nevo R, Kiss V, Grabusic K . Importin 7 and exportin 1 link c-Myc and p53 to regulation of ribosomal biogenesis. Mol Cell. 2012; 45(2):222-32. PMC: 3270374. DOI: 10.1016/j.molcel.2011.11.022. View

Siegfried N, Busan S, Rice G, Nelson J, Weeks K . RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods. 2014; 11(9):959-65. PMC: 4259394. DOI: 10.1038/nmeth.3029. View

Zhao B, Hu X, Zhou Y, Shi Y, Qian R, Wan Y . Characterization of the aberrant splicing of DVL2 induced by cancer-associated SF3B1 mutation. Biochem Biophys Res Commun. 2021; 546:21-28. DOI: 10.1016/j.bbrc.2021.01.084. View

Darman R, Seiler M, Agrawal A, Lim K, Peng S, Aird D . Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3' Splice Site Selection through Use of a Different Branch Point. Cell Rep. 2015; 13(5):1033-45. DOI: 10.1016/j.celrep.2015.09.053. View

Kumar J, Lackey L, Waldern J, Dey A, Mustoe A, Weeks K . Quantitative prediction of variant effects on alternative splicing in using endogenous pre-messenger RNA structure probing. Elife. 2022; 11. PMC: 9236610. DOI: 10.7554/eLife.73888. View

Wang E, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C . Alternative isoform regulation in human tissue transcriptomes. Nature. 2008; 456(7221):470-6. PMC: 2593745. DOI: 10.1038/nature07509. View

Wan Y, Wu C . SF3B1 mutations in chronic lymphocytic leukemia. Blood. 2013; 121(23):4627-34. PMC: 3674664. DOI: 10.1182/blood-2013-02-427641. View

Bubenik J, Hale M, McConnell O, Wang E, Swanson M, Spitale R . RNA structure probing to characterize RNA-protein interactions on a low abundance pre-mRNA in living cells. RNA. 2020; . PMC: 7901844. DOI: 10.1261/rna.077263.120. View

Deigan K, Li T, Mathews D, Weeks K . Accurate SHAPE-directed RNA structure determination. Proc Natl Acad Sci U S A. 2008; 106(1):97-102. PMC: 2629221. DOI: 10.1073/pnas.0806929106. View

10.

Herbert A, Hatfield A, Lackey L . How does precursor RNA structure influence RNA processing and gene expression?. Biosci Rep. 2023; 43(3). PMC: 9977717. DOI: 10.1042/BSR20220149. View

11.

Zamft B, Bintu L, Ishibashi T, Bustamante C . Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases. Proc Natl Acad Sci U S A. 2012; 109(23):8948-53. PMC: 3384149. DOI: 10.1073/pnas.1205063109. View

12.

Wang Y, Xie Z, Kutschera E, Adams J, Kadash-Edmondson K, Xing Y . rMATS-turbo: an efficient and flexible computational tool for alternative splicing analysis of large-scale RNA-seq data. Nat Protoc. 2024; 19(4):1083-1104. DOI: 10.1038/s41596-023-00944-2. View

13.

Van Nostrand E, Freese P, Pratt G, Wang X, Wei X, Xiao R . A large-scale binding and functional map of human RNA-binding proteins. Nature. 2020; 583(7818):711-719. PMC: 7410833. DOI: 10.1038/s41586-020-2077-3. View

14.

Hastings M, Krainer A . Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol. 2001; 13(3):302-9. DOI: 10.1016/s0955-0674(00)00212-x. View

15.

Pan Q, Shai O, Lee L, Frey B, Blencowe B . Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008; 40(12):1413-5. DOI: 10.1038/ng.259. View

16.

Saldi T, Riemondy K, Erickson B, Bentley D . Alternative RNA structures formed during transcription depend on elongation rate and modify RNA processing. Mol Cell. 2021; 81(8):1789-1801.e5. PMC: 8052309. DOI: 10.1016/j.molcel.2021.01.040. View

17.

Wilkinson M, Charenton C, Nagai K . RNA Splicing by the Spliceosome. Annu Rev Biochem. 2019; 89:359-388. DOI: 10.1146/annurev-biochem-091719-064225. View

18.

Zhang Q, Ai Y, Abdel-Wahab O . Molecular impact of mutations in RNA splicing factors in cancer. Mol Cell. 2024; 84(19):3667-3680. PMC: 11455611. DOI: 10.1016/j.molcel.2024.07.019. View

19.

Wilkinson K, Vasa S, Deigan K, Mortimer S, Giddings M, Weeks K . Influence of nucleotide identity on ribose 2'-hydroxyl reactivity in RNA. RNA. 2009; 15(7):1314-21. PMC: 2704086. DOI: 10.1261/rna.1536209. View

20.

Kesarwani A, Ramirez O, Gupta A, Yang X, Murthy T, Minella A . Cancer-associated SF3B1 mutants recognize otherwise inaccessible cryptic 3' splice sites within RNA secondary structures. Oncogene. 2016; 36(8):1123-1133. PMC: 5311031. DOI: 10.1038/onc.2016.279. View