Ribonomic Analysis for Transdifferentiation of Adult Muscle Stem Cell, C2Cl2

Objectives: Emerging technologies using stem cells have various advantages over conventional medical approaches especially for the field of stem cell manipulation to treat severe genetic diseases. We applied recently developed ribonomic analysis to dissect molecular mechanism for differentiation of adult muscle stem cell to myotube. Ribonomics, an advanced transcriptomic tool, utilized specific RNA-protein complexes to reveal cellular changes during environmental fluctuations such as differentiation. This method can be more powerful when gene expressions governed mainly by RNA turnover are analyzed. We generate monoclonal antibodies against HuR to study the role of HuR-specific RNA-protein complexes during muscle differentiation or transdifferentiation into adipocyte. Methods: In order to establish ribonomic approaches, we had generated polyclonal or monoclonal antibodies against HuR, a regulatory protein for RNA turnover. RNA gel shift, supershift, and RNA turnover assay for RNA degradation was also utilized to analyze adult stem cell differentiation. As a model system for adult stem cell differentiation, C2C12 cell line or primary satellite cell was tested for myotube formation. Results: Our monoclonal antibody against HuR was very specific based on Western blot using various recombinant protein or cell lysate. Also this antibody was capable of immunoprecipiting endogenous HuR proteins and detect HuR proteins from cell lines or sectioned tissue. For RNA analysis, this antibody supershifted RNA probe bound by recombinant HuR or cell lysate. RNA decay assay was also established using RAW cells and KB, an oral epithelial cell line. During the differentiation of C2C12, immunofluoresence by HuR antibody revealed that HuR was translocated from nucleus to cytoplasm in which HuR was known to stabilize its target RNA. Conclusion: Newly generated HuR-specific monoclonal antibody will be useful to study the regulation of HuR-specific RNA involved in differentiation or transdifferentiation of muscle stem cell.