Further Characterization of a Novel Microtubule-Binding Protein CLIPR-76 and Its Role in Cardiomyocyte Development

CLIPR-76 is a formally uncharacterized protein capable of binding both microtubules (MTs) and membranes. Encoded by the CLIP4 gene, human CLIPR-76 consists of 15 exons spanning 166 kb with the longest open reading frame encoding a 76 kDa protein. Foundational work in the lab has demonstrated that exogenously expressed isoforms have alternative localizations, with isoforms 1, 2, and 3 localizing to MTs while isoform 4 localizes to the endoplasmic reticulum. Previously, our lab has demonstrated that CLIP4 mRNA is most highly expressed in striated muscle and CLIPR-76 protein is upregulated when murine C2C12 myoblast cells are induced to differentiate into skeletal muscle. Taken together, these observations suggest CLIPR-76 plays a role in the development of striated muscle, perhaps by helping to direct the cytoskeletal and ER/sarcoplasmic reticulum membrane rearrangements necessary for myogenesis. Here we utilize a combination of cell and molecular biological techniques as well as bioinformatic and biochemical analyses to further study CLIPR-76 and its role in striated muscle development, particularly cardiomyocyte development from human induced pluripotent stem cells (iPSCs). Updated sequence analysis indicates that CLIPR-76 has additional isoforms than previously estimated and, while the gene and protein are remarkably conserved across species, there is evidence of different methods of alternative splicing utilized between human and mouse genomes. Immunofluorescence studies demonstrate that CLIPR-76 expression and localization change during both skeletal and cardiac muscle development. Alterations to the CLIP4 gene using CRISPR editing is correlated to perturbation in the expected developmental timeline of iPSC-derived cardiomyocytes, suggesting a role in early cardiac muscle development.