Abstract Thu111: The splicing factor SF3B1 in regulating endothelial gene expression and angiogenesis

Endothelial cells (EC) that line the innermost layer of blood vessels play fundamental roles in the cardiovascular system in both development and diseases. Research in the past 30 years revealed key roles of signaling pathways and their downstream transcriptional events in governing EC gene expression and function. But post-transcriptional regulation by RNA-binding proteins and splicing factors are also critical for gene expression. Yet, a key knowledge gap in vascular biology resides in our understanding of the roles of splicing factors in regulating EC function via post-transcriptional regulation. From combined in silico (single-cell RNAseq) and in vitro screens, we identified the splicing factor SF3B1 as the top candidate whose knockdown completely abrogated angiogenesis and led to unhealthy-looking EC. SF3B1 is a core structural component of spliceosome and frequently mutated in various types of cancer. With RNAseq, we further found that SF3B1 knockdown in vitro caused mis-splicing of over 30% of the transcriptome, which resulted in abolished EC proliferation and increased cell death. Pathway analysis of genes that are both mis-spliced and differentially expressed revealed enrichment of cell cycle genes among down-regulated genes and enrichment of ferroptosis genes among those upregulated. We then utilized a new Sf3b1 floxed mouse line and determined how EC-specific knockout (iECKO) of Sf3b1 impacted on angiogenesis in vivo . Analysis of the P4 retinas of Sf3b1 iECKO pups showed significantly impaired angiogenesis characterized by extremely low EC density and EC proliferation. Our data here show for the first time that SF3B1 is required for EC proliferation and survival, implicating an essential role of SF3B1 in EC gene expression and function. Because anti-cancer drugs targeting SF3B1 and other spliceosome mutations are under active clinical trials, our data here also suggest that vascular toxicity needs to be considered in future therapies.