KH1 Domain of Poly C Binding Protein 1 (PCBP1) Interacts with Utrophin-A mRNA and Modulates Utrophin-A Expression in Mouse Myoblasts

Abstract

Utrophin upregulation is an attractive strategy to ameliorate Duchenne Muscular Dystrophy (DMD). Here, we investigated whether the murine poly(C)-binding protein 1 (PCBP1) directly interacts with the utrophin-A mRNA, and how the interaction shapes the fate of utrophin-A in muscle cells. As no experimentally validated structure of the murine PCBP1 is available, AlphaFold2 was utilized to predict the structure of the full-length protein and the KH1-KH2 domain of the protein. The more stable and reliable model of the KH1-KH2 domain of PCBP1 was then docked with the 5'UTR of the utrophin-A mRNA, and the top-ranked docked complex was utilized for MD simulation to identify the most stable hydrogen bond-forming amino acid residues from PCBP1. ITC showed a thermodynamically favorable spontaneous interaction between the full-length PCBP1 and the 5'UTR of utrophin-A mRNA, but the deletion mutant of PCBP1, lacking the predicted contact region, failed to exhibit any interaction. This observation was also confirmed by an RNA-immunoprecipitation assay in C2C12 cells. However, the mutant PCBP1 was unable to alter the expression of the utrophin-A in the C2C12 cells. In cells, PCBP1 overexpression lowered utrophin-A expression, while shRNA knockdown produced reciprocal increases. A 5'UTR-luciferase reporter mirrored this observation. Finally, subcellular fractionation showed that PCBP1 promotes nuclear retention of utrophin-A transcripts and reduces their cytoplasmic availability, with reciprocal shifts upon PCBP1 knockdown. Considering earlier work on PCBP2, our data suggest both paralogs recognize the same 5'UTR element, and the KH1-dependent interaction represses utrophin-A by coupling nuclear retention to reduced protein output. Thus, the PCBP1-5'UTR interaction emerges as a potent target for utrophin de-repression in DMD.