RNA binding proteins RBPMS and RBPMS2 regulate alternative splicing and act in ribonucleoprotein granule formation

Abstract

The study reveals that cardiomyocyte (CM) specific ablation of RBPMS and RBPMS2 (RBPMS/2) results in embryonic lethality in conjunction with cardiac malformation. The analysis demonstrates that RBPMS/2 promote alternative splicing (AS), which are essential for heart development and cardiomyocyte identity. Interestingly, overexpression of the dominant isoform RBPMSA induces cardiomyocyte-specific alternative splicing in several non-cardiac tissues. The network of alternative splicing regulated by RBPMS/2 predominantly controls organization and function of the cytoskeleton and sarcomere apparatus, in particular components of M-band and Z-disc, as well as calmodulin binding. Intriguingly, RBPMS/2 deficient cardiomyocytes show multipolar spindle defects and chromosomal mis-segregation, which is causative for an increased frequency of nuclear abnormalities. Furthermore, the study has unraveled that inactivation of a cardiomyocyte specific splicing variant of Camk2g, a specific target of RBPMS, results in spindle defects, resembling abnormalities observed in RBPMS/2 double knock out (dKO) cardiomyocytes. In conclusion, the study indicates that RBPMS and RBPMS2 co-regulate alternative splicing in a developmental-stage-specific manner to enable proper sarcomere apparatus and spindle formation. In addition to the function of RBPMS/2 in alternative splicing, RBPMS operates in the regulation of processing bodies (PBs) and stress granules (SGs) formation in cytoplasm of cardiomyocytes. An interactome screen based on GFP-tagged RBPMS identified interaction partners of the splicing and the RNA stability pathway. Under normal and stress conditions, RBPMS co-localizes with typical RNP-granule markers in the cytoplasm of cardiomyocyte. Inactivation of RBPMS/2 in embryonic cardiomyocyte under physiological conditions results in giant PBs. In contrast, RBPMS/2 deficient embryonic cardiomyocytes fail to form SGs under stress conditions. Furthermore, the formation of RBPMS containing RNP-granules was massively disturbed in DCP1A deficient cardiomyocytes. These results obtained during the course of the study provide first insights in dynamic RNP-granule formation in cardiomyocytes under physiological and pathophysiological conditions.