CRISPR/Cas9 screening of RNA binding proteins (RBPs) that regulate RUNX1 isoform production

The proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined, central role and mutations promote malignant transformation. More recently, studies have illuminated the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between post-transcriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the post-transcriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a versus RUNX1b/c) that mediate HSC self-renewal versus differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detecting changes in RNA-processing. We utilized this reporter in a CRISPR screen consisting of single guide-RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the post-transcriptional regulation of a key hematopoietic transcription factor and identifies RBPs which may have a widespread, important function in hematopoiesis. CRISPR screen of enriched sgRNAs targeting RBPs in GFP high and low cell populations compared to the bulk cell population at day 21, by sequencing of gDNA in biological duplicates. Day 0 gDNA sequencing in biological duplicates was also compared to day 21. Also included are 3'RNA sequencing data from three biological replicates of healthy, human common myeloid progenitors (CMPs) isolated from leukapheresis products. Bigwig files were generated to visualize poly(A) site usage of the RUNX1 gene.