RBPMS and RBPMS2 Cooperate to Safeguard Cardiac Splicing

Abstract Background Mutations in cardiac splicing factors (SFs) cause cardiomyopathy and congenital heart disease, underscoring the critical role of SFs in cardiac development and disease. Cardiac SFs are implicated to cooperatively regulate the splicing of essential cardiac genes, but the functional importance of their collaboration remains unclear. RNA Binding Protein with Multiple Splicing (RBPMS) and RBPMS2 are SFs involved in heart development and exhibit similar splicing regulatory activities in vitro , but it is unknown whether they cooperate to regulate splicing in vivo . Methods Rbpms and Rbpms2 single or double cardiomyocyte (CM)-specific knockout (KO) mice were generated and analyzed for cardiac phenotypes. RNA sequencing was performed to assess gene expression and splicing changes in single and double KOs. In silico analyses were used to dissect the mechanisms underlying distinct and overlapping roles of RBPMS and RBPMS2 in heart development. Results Mice lacking both RBPMS and RBPMS2 in CMs died before embryonic day 13.5 and developed sarcomere disarray, whereas Rbpms or Rbpms2 single CM-specific KO mice had normal sarcomere assembly and survived to adulthood. Defective sarcomere assembly is likely owing to the widespread mis-splicing of genes essential for cardiac contraction in double KO mice, underscoring the overlapping role of RBPMS and RBPMS2 in splicing regulation. Mechanistically, we found RBPMS and RBPMS collectively promote cardiac splicing program while repressing non-cardiac splicing programs. Moreover, RNA splicing maps suggested that the binding location of RBPMS and RBPMS2 on pre-mRNA dictates whether they function as splicing activators or repressors. Lastly, the requirement for RBPMS and/or RBPMS2 for splicing regulation arises from intrinsic features of the target exons. Conclusions Our results demonstrate that RBPMS and RBPMS2 work in concert to safeguard the splicing of genes essential for cardiac contraction, highlighting the importance of SF collaboration in maintaining cardiac splicing signature, which should be taken into consideration when devising future therapeutic approaches through modulating the activity of SFs. Novelty and Significance What Is Known? Mutations in cardiac splicing factors (SFs) cause cardiomyopathy and congenital heart disease, and the splicing of cardiac genes is regulated by multiple SFs. However, the functional importance of the collaboration among specific cardiac SFs is unknown. RBPMS has emerged as a cardiac SF for sarcomere genes but is not required for sarcomere assembly. RBPMS2 can substitute RBPMS in in vitro splicing assays, yet its role in mammalian cardiomyocytes (CMs) remains unclear. What New Information Does This Article Contribute? RBPMS and RBPMS2 have both distinct and overlapping roles in CMs. RBPMS and RBPMS2 collectively contribute to the maintenance of cardiac splicing program, which is essential for sarcomere assembly and embryonic survival. RNA splicing map of RBPMS and RBPMS2 reveals that they can function either as splicing activators or repressors, depending on their binding locations on pre-mRNA. This study provides compelling evidence of cooperation between cardiac splicing factors during heart development, which, to our knowledge, has not been demonstrated in vivo . Rbpms and Rbpms2 CM-specific double KO mice die in utero and exhibit sarcomere disarray, whereas single KO mice survive to adulthood with normal sarcomere structure but manifest distinct cardiac phenotypes, suggesting RBPMS and RBPMS2 possess both distinct and overlapping functions in CMs. Although mis-splicing in cardiac genes can be seen in all three KOs, the splicing signature of double KO hearts drastically shifts towards non-cardiac tissues, including more prominent mis-splicing in genes related to cardiac contractile function. Our study further reveals that the splicing regulation of RBPMS and RBPMS2 has the characteristics of “positional effects”, i.e., the binding location on pre-mRNA dictates whether they function as splicing activators or repressors; and the intrinsic features of the target exon determine the requirement for one or two RBPMS proteins for splicing regulation. Our study sheds light on the functional importance of cardiac SF cooperation in maintaining cardiac splicing signature during heart development.