RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.

Abstract

RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. We performed a differential expression screen in zebrafish embryos to identify genes enriched in <i>nkx2.5</i>-positive cardiomyocytes or cardiopharyngeal progenitors compared to <i>nkx2.5</i>-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [<i>RBPMS2</i>)] by generating and characterizing <i>rbpms2</i> knockout zebrafish and human cardiomyocytes derived from <i>RBPMS2</i>-deficient induced pluripotent stem cells. We identified 1848 genes enriched in the <i>nkx2.5</i>-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs <i>rbpms2a</i> and <i>rbpms2b</i>, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require <i>RBPMS2</i> for alternative splicing regulation, including <i>RBFOX2, SLC8A1</i>, and <i>MYBPC3</i>. Our study identifies <i>RBPMS2</i> as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.