Functional Characterization of RBPMS-Positive Ribonucleoprotein Granules in Cardiomyocytes

Abstract

RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of the RNA-binding protein family and localizes to both the nucleus and cytoplasm. In addition to its function in transcriptional co-regulation, RBPMS may also play a role in the cytoplasm for regulation of mRNA stability, transport and recruitment into cytoplasmic granules. Although aberrant RNA processing and dysregulated ribonucleoprotein assemblies are known to contribute to the pathogenesis of neurodegenerative diseases, the composition of RNPs and their (patho)physiological roles in the cardiovascular system remain poorly understood. In this study, I investigated the role of RBPMS in regulating RNP assemblies and their dynamics in response to cardiovascular stress. I developed an optimized protocol for APEX2-mediated proximity labeling in murine HL-1 cardiomyocytes. The approach enabled high-resolution mapping of the cardiac stress granule proteome, revealing a previously uncharacterized complexity and tissue-specific adaptation in SG composition. Interestingly, proximity interactions between SG components in unstressed cells point to pre-assembly mechanisms facilitating rapid SG formation during stress. Interactome analysis of GFP-tagged RBPMSA and RBPMSB isoforms identified key partners involved in DNA damage response and phase separation. Additionally, RBPMSB was found to interact with mitochondrial proteins, potentially linking phase-separated compartments and cellular organelles in stress responses. Upon doxorubicin-induced DNA damage, RBPMS initially translocates to SGs in the cytoplasm and subsequently forms nuclear puncta, coinciding with chromatin reorganization. These observations suggest that RBPMS contributes to both acute and long-term stress adaptation, potentially modulating DDR and chromatin phase transitions. Importantly, doxorubicin-induced genotoxic stress alters the abundance and localization of RBPMS isoforms, disrupts splicing factor dynamics, and impairs mitochondrial biogenesis, collectively contributing to cardiotoxicity. The identification of age-dependent differences in alternative splicing regulation further highlights the complexity of the cardiac stress response. The interplay between RBPMS, splicing factors, and SGs emerged as a critical determinant of cardiomyocyte survival and function under stress. Taken together, my findings establish RBPMS as a regulator of RNP assembly and cellular stress responses in cardiovascular disease, highlighting its potential as a therapeutic target and providing a foundation for further investigation into its role in DNA damage response and stress-related cardiac pathologies.