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Cancer-associated SF3B1 mutants recognize otherwise inaccessible cryptic 3′ splice sites within RNA secondary structures

Oncogene volume 36, pages 1123–1133 (2017)Cite this article

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Abstract

Recurrent mutations in core splicing factors have been reported in several clonal disorders, including cancers. Mutations in SF3B1, a component of the U2 splicing complex, are the most common. SF3B1 mutations are associated with aberrant pre-mRNA splicing using cryptic 3′ splice sites (3′SSs), but the mechanism of their selection is not clear. To understand how cryptic 3′SSs are selected, we performed comprehensive analysis of transcriptome-wide changes to splicing and gene expression associated with SF3B1 mutations in patient samples as well as an experimental model of inducible expression. Hundreds of cryptic 3′SS were detectable across the genome in cells expressing mutant SF3B1. These 3′SS are typically sequestered within RNA secondary structures and poorly accessible compared with their corresponding canonical 3′SS. We hypothesized that these cryptic 3′SS are inaccessible during normal splicing catalysis and that this constraint is overcome in spliceosomes containing mutant SF3B1. This model of secondary structure-dependent selection of cryptic 3′SS was found across multiple clonal processes associated with SF3B1 mutations (myelodysplastic syndrome and chronic lymphocytic leukemia). We validated our model predictions in mini-gene splicing assays. Additionally, we found deregulated expression of proteins with relevant functions in splicing factor-related diseases both in association with aberrant splicing and without corresponding splicing changes. Our results show that SF3B1 mutations are associated with a distinct splicing program shared across multiple clonal processes and define a biochemical mechanism for altered 3′SS choice.

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Acknowledgements

Work was funded in part by the National Institutes of Health (R01 HL104070 to MMP and R01 HL098608 to ACM). We thank Barry Paw (Boston Children's Hospital, Boston, MA, USA) for anti-Mitoferrin antibody, Thomas A Cooper (Baylor College of Medicine, Houston, TX, USA) for the RG6 plasmid and Karla Neugebauer (Yale University) for helpful suggestions. We also thank The Yale Center for Genome Analysis (YCGA) for high-throughput sequencing and the Yale University Biomedical High Performance Computing Center for use of compute clusters to run bioinformatics analysis.

Author contributions

AK analyzed next-generation data and wrote the manuscript; OR performed experiments and wrote the manuscript; XY, AG and TM performed experiments; ACM designed some experiments, analyzed the data and edited the manuscript; MMP initiated the study, performed experiments, analyzed the data, provided supervision and wrote the manuscript with input from other authors.

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  1. A K Kesarwani and O Ramirez: These authors contributed equally to this work.

Authors and Affiliations

  1. Section of Hematology, Yale Cancer Center and Yale University School of Medicine, New Haven, CT, USA

    A K Kesarwani, O Ramirez, A K Gupta, X Yang & M M Pillai

  2. Driskill Graduate Program, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    T Murthy

  3. Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA

    A C Minella

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  1. A K Kesarwani
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Correspondence to M M Pillai.

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Kesarwani, A., Ramirez, O., Gupta, A. et al. Cancer-associated SF3B1 mutants recognize otherwise inaccessible cryptic 3′ splice sites within RNA secondary structures. Oncogene 36, 1123–1133 (2017). https://doi.org/10.1038/onc.2016.279

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