Large-scale Analysis of Branchpoint Usage Across Species and Cell Lines

Overview

Journal Genome Res

Specialty Genetics

Date 2017 Jan 26

PMID 28119336

Citations 74

Authors

Allison J Taggart

Chien-Ling Lin

Barsha Shrestha

Claire Heintzelman

Seongwon Kim

William G Fairbrother

Affiliations

Soon will be listed here.

Abstract

The coding sequence of each human pre-mRNA is interrupted, on average, by 11 introns that must be spliced out for proper gene expression. Each intron contains three obligate signals: a 5' splice site, a branch site, and a 3' splice site. Splice site usage has been mapped exhaustively across different species, cell types, and cellular states. In contrast, only a small fraction of branch sites have been identified even once. The few reported annotations of branch site are imprecise as reverse transcriptase skips several nucleotides while traversing a 2-5 linkage. Here, we report large-scale mapping of the branchpoints from deep sequencing data in three different species and in the SF3B1 K700E oncogenic mutant background. We have developed a novel method whereby raw lariat reads are refined by U2snRNP/pre-mRNA base-pairing models to return the largest current data set of branchpoint sequences with quality metrics. This analysis discovers novel modes of U2snRNA:pre-mRNA base-pairing conserved in yeast and provides insight into the biogenesis of intron circles. Finally, matching branch site usage with isoform selection across the extensive panel of ENCODE RNA-seq data sets offers insight into the mechanisms by which branchpoint usage drives alternative splicing.

Citing Articles

Splicing accuracy varies across human introns, tissues, age and disease.

Garcia-Ruiz S, Zhang D, Gustavsson E, Rocamora-Perez G, Grant-Peters M, Fairbrother-Browne A Nat Commun. 2025; 16(1):1068.

PMID: 39870615 PMC: 11772838. DOI: 10.1038/s41467-024-55607-x.

Circular RNA Formation and Degradation Are Not Directed by Universal Pathways.

Srinivasan A, Mroczko-Mlotek E, Wojciechowska M Int J Mol Sci. 2025; 26(2).

PMID: 39859439 PMC: 11766002. DOI: 10.3390/ijms26020726.

SF3B1: from core splicing factor to oncogenic driver.

Bak-Gordon P, Manley J RNA. 2025; 31(3):314-332.

PMID: 39773890 PMC: 11874996. DOI: 10.1261/rna.080368.124.

Cancer-associated SF3B1 mutation K700E causes widespread changes in U2/branchpoint recognition without altering splicing.

Damianov A, Lin C, Zhang J, Manley J, Black D bioRxiv. 2024; .

PMID: 39605567 PMC: 11601671. DOI: 10.1101/2024.11.18.624191.

Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers.

Yao J, Xu H, Ferrick-Kiddie E, Nottingham R, Wu D, Ares Jr M PLoS Genet. 2024; 20(9):e1011416.

PMID: 39325823 PMC: 11460701. DOI: 10.1371/journal.pgen.1011416.

References

Hall K, Green M, Redfield A . Structure of a pre-mRNA branch point/3' splice site region. Proc Natl Acad Sci U S A. 1988; 85(3):704-8. PMC: 279623. DOI: 10.1073/pnas.85.3.704. View

Smith C, Chu T, Nadal-Ginard B . Scanning and competition between AGs are involved in 3' splice site selection in mammalian introns. Mol Cell Biol. 1993; 13(8):4939-52. PMC: 360135. DOI: 10.1128/mcb.13.8.4939-4952.1993. View

Imielinski M, Berger A, Hammerman P, Hernandez B, Pugh T, Hodis E . Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell. 2012; 150(6):1107-20. PMC: 3557932. DOI: 10.1016/j.cell.2012.08.029. View

Padgett R . New connections between splicing and human disease. Trends Genet. 2012; 28(4):147-54. PMC: 3319163. DOI: 10.1016/j.tig.2012.01.001. View

Corrionero A, Minana B, Valcarcel J . Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A. Genes Dev. 2011; 25(5):445-59. PMC: 3049286. DOI: 10.1101/gad.2014311. View

Taggart A, DeSimone A, Shih J, Filloux M, Fairbrother W . Large-scale mapping of branchpoints in human pre-mRNA transcripts in vivo. Nat Struct Mol Biol. 2012; 19(7):719-21. PMC: 3465671. DOI: 10.1038/nsmb.2327. View

Tseng C, Cheng S . Both catalytic steps of nuclear pre-mRNA splicing are reversible. Science. 2008; 320(5884):1782-4. DOI: 10.1126/science.1158993. View

Bonnal S, Vigevani L, Valcarcel J . The spliceosome as a target of novel antitumour drugs. Nat Rev Drug Discov. 2012; 11(11):847-59. DOI: 10.1038/nrd3823. View

Hoskins A, Moore M . The spliceosome: a flexible, reversible macromolecular machine. Trends Biochem Sci. 2012; 37(5):179-88. PMC: 3508674. DOI: 10.1016/j.tibs.2012.02.009. View

10.

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

11.

Katz Y, Wang E, Airoldi E, Burge C . Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods. 2010; 7(12):1009-15. PMC: 3037023. DOI: 10.1038/nmeth.1528. View

12.

Awan A, Manfredo A, Pleiss J . Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans. Proc Natl Acad Sci U S A. 2013; 110(31):12762-7. PMC: 3732940. DOI: 10.1073/pnas.1218353110. View

13.

Libri D, dAubenton-Carafa Y, Fiszman M, BRODY E, Marie J . In vitro splicing of mutually exclusive exons from the chicken beta-tropomyosin gene: role of the branch point location and very long pyrimidine stretch. EMBO J. 1990; 9(1):241-9. PMC: 551653. DOI: 10.1002/j.1460-2075.1990.tb08101.x. View

14.

Alsafadi S, Houy A, Battistella A, Popova T, Wassef M, Henry E . Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage. Nat Commun. 2016; 7:10615. PMC: 4743009. DOI: 10.1038/ncomms10615. View

15.

Alioto T . U12DB: a database of orthologous U12-type spliceosomal introns. Nucleic Acids Res. 2006; 35(Database issue):D110-5. PMC: 1635337. DOI: 10.1093/nar/gkl796. View

16.

Shao C, Yang B, Wu T, Huang J, Tang P, Zhou Y . Mechanisms for U2AF to define 3' splice sites and regulate alternative splicing in the human genome. Nat Struct Mol Biol. 2014; 21(11):997-1005. PMC: 4429597. DOI: 10.1038/nsmb.2906. View

17.

Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A . Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495(7441):333-8. DOI: 10.1038/nature11928. View

18.

Smith C, Nadal-Ginard B . Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing. Cell. 1989; 56(5):749-58. DOI: 10.1016/0092-8674(89)90678-8. View

19.

Mercer T, Clark M, Andersen S, Brunck M, Haerty W, Crawford J . Genome-wide discovery of human splicing branchpoints. Genome Res. 2015; 25(2):290-303. PMC: 4315302. DOI: 10.1101/gr.182899.114. View

20.

Gao K, Masuda A, Matsuura T, Ohno K . Human branch point consensus sequence is yUnAy. Nucleic Acids Res. 2008; 36(7):2257-67. PMC: 2367711. DOI: 10.1093/nar/gkn073. View