Identification and characterization of RBM12 as a novel regulator of fetal hemoglobin expression

Abstract

The fetal-to-adult hemoglobin transition is clinically relevant as reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and β-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1 are known post-transcriptional regulators of HbF production but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RNA Recognition Motif (RRM) domains and identified RBM12 as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from SCD patients with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced crosslinking and immunoprecipitation followed by high throughput sequencing (eCLIP-Seq) revealed strong preferential binding of RBM12 to 5'UTRs of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of five RRM domains as essential, and, in conjunction with a linker domain, as sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and β-thalassemia.

The psychosis risk factorRBM12encodes a novel repressor of GPCR/cAMP signal transduction

“…Studies done in primary human erythroblasts, A-431, and U-2OS cells found that RBM12 displayed nuclear localization. 9,25 In agreement with these reports, we observed nuclear localization for native RBM12 under both basal and isoproterenol-induced conditions in HEK293 cells (Fig. 4b).…”

“…RBM12 is predominantly localized to the nucleus 25 and has been shown to bind RNA and to tentatively impact transcript splicing. 9 Based on the present work, we cannot rule out that the cytosolic fraction of RBM12 plays a direct regulatory role in GPCR signaling. Studies aimed at defining the RBM12 interactome could resolve this possibility.…”

“…A recent study found a novel role of RBM12 as a repressor of fetal hemoglobin expression. 9 Notably, the authors reported a requirement only for the N-terminal portion of the protein in that process, whereas deletion of the C-terminal RRM domain, which is truncated in the psychosis cohorts, was dispensable from that regulation. Therefore, the C-terminal RRM domain and its interactome must be of particular significance in the context of RBM12 and its role in GPCR/cAMP signaling.…”

“…Interestingly, RBM12 has no known ties to GPCR biology. It is annotated as an RNA-binding protein (RBP) based on co-immunoprecipitation with RNA 9 and based on its amino acid sequence, which is predicted to contain three RNA-recognition motifs (RRMs). 10 In terms of physiology, RBM12 was recently discovered as a high-penetrance risk factor for familial schizophrenia and psychosis in a family-based whole-genome study to identify rare coding-sequence variants associated with disease segregation in a pedigree.…”

“…12 Outside of its functions in the brain, RBM12 was found to be a repressor of fetal hemoglobin expression. 9 Therefore, RBM12 is of clear significance for a range of physiologies. Yet, the cellular functions and pathways to which RBM12 belongs are unknown, and therefore it remains unclear how mutations in this gene contribute to disease pathobiology.…”