Background: Mutations in cardiac SFs (splicing factors) 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. RBPMS (RNA-binding protein with multiple splicing) and RBPMS2 (RNA-binding protein with multiple splicing 2) 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-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 and minigene splicing assays were used to dissect the mechanisms underlying the roles of RBPMS and RBPMS2 in heart development.
Results: Mice lacking both RBPMS and RBPMS2 in cardiomyocytes died before embryonic day 13.5 and developed sarcomere disarray, whereas Rbpms or Rbpms2 single-cardiomyocyte-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 that RBPMS and RBPMS2 collectively promote cardiac splicing programs while repressing noncardiac splicing programs. Moreover, RNA splicing maps suggested that the binding location of RBPMS and RBPMS2 on precursor messenger RNA dictates whether they function as splicing activators or repressors. Last, the sensitivity to the dosage of RBPMS and 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.
Keywords: RNA splicing; RNA splicing factors; alternative splicing; cardiomyopathies; myocytes, cardiac; sarcomeres.