Project description:Revealing the binding sites of endogenous SF3B1 using HITS-CLIP (high-throughput sequencing coupled with cross-linking immunoprecipitation).
Project description:SF3B1 is one of shared components of U2 and U12 snRNPs that are required for splicing of U2-type and U12-type introns, respectively. Interestingly, recurrent somatic mutations have been identified in the spliceosome components in human cancer. By using CLIP-seq for SF3B1, binding sites in human embryonic stem cells were determined. The most prominent binidng sites were at 3' splice sites on pre-mRNA. Interestingly, 5' and 3' ends of introns spliced out were enriched by CLIP.
Project description:Alternative splicing plays a critical role in generating transcriptome diversity, and aberrant splicing is frequently observed in cancer. Mutations in the splicing factor SF3B1 are particularly common in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS), with different survival prognoses. We applied long-read sequencing for the investigation of the SF3B1 mutation effect on the transcriptome of MDS and CLL patients, as well as isogenic cell lines. Our results revealed that SF3B1 mutation effect was common across the different diseases and specifically altered the usage of 3’ alternative splice sites within short proximity to the canonical splice sites. Based on computational simulations, the most common K700E mutation led to destabilization of the SF3B1-mRNA binding. Furthermore, we combined the full isoform information with genome-wide SF3B1-RNA binding maps to predict the functional consequences of the aberrant splicing and gain mechanistic insights into the role of mutated SF3B1 in splicing.