Long-read sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations in cancer (SF3B1 & U2AF2 iCLIP)
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. Overall design: To understand the RNA binding behavior of SF3B1, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to map SF3B1 binding sites throughout the transcriptome. For this, we immunoprecipitated SF3B1 from K562-SF3B1wt/wt and K562-SF3B1K700E/wt cells,