Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations [TRIBE-seq]

Mutations in the RNA splicing factor SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing but therapeutic means to correct this mis-splicing do not exist. Here we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SUGP1, a G-patch protein recently implicated in SF3B1 mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1 splicing defects and was sufficient to improve hematopoiesis in SF3B1 mutant mouse and human cells. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and the therapeutic impact of correcting aberrant splicing in SF3B1 mutant cancers. To determine the RNA binding sites of GPATCH8 and SUGP1 transcriptome-wide we performed TRIBE-seq of HEK293T cells with expression of either GPATCH8-ADAR or ADAR-SUGP1 fusion proteins, where ADAR is the catalytic domain of the hyperactive Drosophila adenosine deaminase. After background subtraction of endogenous RNA editing events and editing events created by a control ADAR fusion protein (MCP-ADAR or ADAR-MCP) we identified RNA editing events that were conserved between both replicates of GPATCH8-ADAR or ADAR-SUGP