title: Cancer-associated SF3B1 mutants recognize otherwise inaccessible cryptic 3' splice sites within RNA secondary structures.
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.
authors.affiliation: Section of Hematology, Yale Cancer Center and Yale University School of Medicine, New Haven, CT, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Kesarwani
authors.affiliation: Section of Hematology, Yale Cancer Center and Yale University School of Medicine, New Haven, CT, USA.
authors.forename: O
authors.initials: O
authors.lastname: Ramirez
authors.affiliation: Section of Hematology, Yale Cancer Center and Yale University School of Medicine, New Haven, CT, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Gupta
authors.affiliation: Section of Hematology, Yale Cancer Center and Yale University School of Medicine, New Haven, CT, USA.
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.affiliation: Driskill Graduate Program, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: T
authors.initials: T
authors.lastname: Murthy
authors.affiliation: Blood Research Institute, BloodCenter of Wisconsin, Milwaukee WI, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Minella
authors.affiliation: Section of Hematology, Yale Cancer Center and Yale University School of Medicine, New Haven, CT, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Pillai
mesh_terms: D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D009190:Myelodysplastic Syndromes / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D000072260:RNA Splicing Factors / Q000235:genetics*; D012334:RNA, Neoplasm / Q000737:chemistry* / Q000235:genetics; D017381:Spliceosomes; D059467:Transcriptome; D014407:Tumor Cells, Cultured
chemical_list: D010750:Phosphoproteins; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012334:RNA, Neoplasm; C112491:SF3B1 protein, human