Transcriptome-wide analysis of RNA binding and splicing alterations induced by SRSF2 P95 mutations in myelodysplasia

Mutations in the splicing factor Serine Arginine Rich Splicing Factor 2 (SRSF2) occur in over 30% of patients with myelodysplasia (MDS) and a subset of patients with acute myeloid leukemia (AML) and portend a poor prognosis. SRSF2 is ubiquitously expressed and directs exon inclusion or exclusion in a sequence and context dependent manner. SRSF2 mutations almost exclusively affect the proline at position 95 in the C-terminus of the RNA binding domain. We here show how a mutation in SRSF2 (P95H) affects its normal function in vivo on a transcriptome-wide basis via RNA crosslinking and immunoprecipitation (HITS-CLIP) and parallel quantification of transcript levels (RNA-seq), leading to the identification of differentially bound and alternatively spliced SRSF2 targets. Keywords: Splicing, myeloid neoplasia, SRSF2, HITS-CLIP In this study, we performed high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to determine the function of mutant SRSF2 in the context of a human hematopoietic cell line. Human erythroid leukemia (HEL) cell lines were stably transduced with either wild type SRSF2 (WT) or mutant SRSF2 (P95H, MUT). HITS-CLIP was performed in biological quadruplicate. RNA-seq was performed in biological duplicate.