RNA-binding proteins (RBPs) regulate all aspects of post-transcriptional gene expression and are critical determinants of cardiac development and disease. In the heart, RBPs control processes ranging from alternative splicing to local translation and stress adaptation. Beyond canonical RBPs, recent RNA interactome studies have uncovered many non-canonical RBPs (ncRBPs) lacking classical RNA-binding domains, suggesting that reciprocal protein::RNA interactions may represent a widespread regulatory principle.
In a previous RNA interactome study in cardiomyocytes (CMs), we identified numerous previously unrecognized ncRBPs, among them several Z-disc-associated proteins. Z-discs are increasingly recognized as dynamic mechanosensory hubs integrating mechanical signals and growing evidence for RNA localisation to Z-discs calls for functional investigation of Z-disc protein interactions with RNA.
Here, using enhanced RNA interactome capture in human iPSC-derived CMs, we report on the identification of Muscle LIM Protein (MLP/CSRP3) as an evolutionary conserved ncRBP. MLP is a highly conserved Z-disc-associated protein implicated in mechano-transduction with loss of MLP or its homologs causing severe phenotypes across species and pathogenic CSRP3 variants being linked to human cardiomyopathy.
Biochemical domain mapping, integrated with published RBDmap data of the MLP paralogue CSRP1, identified two short motifs at the interface of LIM domains and flexible glycine-rich regions as MLP’s putative RNA binding surfaces and targeted mutation of both regions markedly reduced RNA-binding activity.
Notably, one of these regions overlaps with several disease-associated variants.
To functionally characterise the MLP::RNA-interaction, we employed a multi-omics approach which, however, did not support a major role of MLP in transcript-specific translational regulation.
Instead, co-immunoprecipitation (co-IP) following RNase treatment, RNA-dependent sedimentation profiling (R-DeeP) and co-IP analysis of the RNA-binding deficient (RBdef) mutants collectively suggested a role for RNA-binding in shaping MLP’s cytoskeletal protein interaction network.
Additionally, RBdef mutants exhibited increased extractability in fractionation assays, suggesting their impaired integration into higher-order assemblies.
Together with the reported dynamic behaviour of MLP, the requirement for multiple distinct regions to support RNA association and stable assembly suggests a multivalent interaction mode, challenging models of MLP as a static anchor maintained by high-affinity protein interactions.
Supporting its physiological relevance, MLP’s RNA-binding was dynamically regulated by angiotensin II (Ang II) but not phenylephrine (PE) stimulation. Consistently, siRNA-mediated depletion of MLP selectively impaired efficient anabolic adaptation to Ang II, while PE responses remained largely preserved. Putative contributions of RNA-dependent MLP assemblies are currently tested in rescue experiments using wt and RBdef MLP constructs.
Although the precise molecular mechanisms remain to be closer defined, our findings identify regulated RNA association as a functional property of MLP and suggest that RNA-protein interactions may contribute to the organization of adaptive protein assembly dynamics in CMs. Multiple LIM domain proteins have emerged across RNA interactomes, warranting future investigation into whether RNA-dependent assembly may represent a generic principle of LIM protein biology.
