Fig. 4

RRM and zinc finger domains of RBM5 are essential in AML. a RBM5 protein structure. The illustration shows the major canonical functional domains in the human RBM5 protein. The amino acid distance spanning each functional domain is indicated in the colorful pane below. RNA recognition domain (RRM), zinc-finger domain (ZF), octamer repeat (OCRE) domain, glycine-rich region (G-patch) domain. b CRISPR dropout screen by tilling the sgRNAs targeting the RBM5 exons in OCIAML2 cells at day 7. The known protein domains (including RRM1, RRM2, ZF-RanBP, ZF-C2H2, OCRE, and G-patch) were highlighted in purple boxes, while the predicted essential domains were highlighted in brown boxes. The Z-score was calculated based on the average count of all sgRNAs targeting a 10-kb window in the genome. c Western blot analysis was conducted in OCIAML2 cells transduced with RBM5 cDNA wild-type (WT) or individual mutant (upper panel) to validate the expression of RBM5 (lower panel). The β-ACTIN was used as a reference. d Competition-based proliferation assay in OCIAML2 expressing the indicated individual RBM5 truncated mutant cDNAs, RBM5-wild-type cDNA (WT), RBM5-sgRNA1-resistant mutant cDNA (PAM-MUT), and empty vector (all monitored by Venus reporter), followed by transduction with RBM5 sg1 or non-targeted sgRNA (NT). Data shown are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, unpaired Student’s t-test