Long Noncoding RNAs in Cardiac and Skeletal Muscle Differentiation during Mouse Embryogenesis

Development is a multistep process that involves a close co-ordination of gene regulatory networks. Lineage specification is a crucial step involved in embryogenesis and understanding the significant steps involved in gene regulatory mechanisms is critical. Long non-coding RNAs, longer than 200 nucleotides have been identified as a new regulator of many molecular mechanisms involved in the development and pathological conditions. From the genome-wide transcriptome analysis of Mesp1-lineage reporter mouse ESC line (UH3), we identified mesoderm specific lncRNAs, from which we selected a subset of 12 lncRNAs for functional characterization. Of this, we identified lincRNA Platr14 to have a positive expression in vivo in cardiac plate and somites of E9.5 mouse embryos. Platr14 was seen enriched in vitro in the undifferentiated AB2.2 ES cell line, and the inhibition of Platr14 showed a decrease in the beating percentage of embryoid bodies which correlated with a deregulation of the mesoderm and cardiac-specific genes. Since Platr14 was enriched in the region of the myotome of E9.5 Embryos, we studied its role in myogenesis. Platr14 showed enrichment in skeletal muscle and the tongue in E15.5 embryonic mouse tissues. Knockdown of Platr14 in C2C12 mesenchymal cell line showed a deregulation of myogenic markers as well as a reduction in the fusion rate for the myotube formation. Though overexpression of Platr14 showed an up-regulation of the main myogenic markers, it did not show any significant change in the myotube formation rate. Strikingly, Platr14 overexpression in the terminal stages showed a repression in the expression of terminal myogenic markers. The whole transcriptome analysis by RNA-Sequencing of differentiating C2C12 myoblasts at day 2, showed a down-regulation of genes associated with the mesenchymal formation, somitogenesis, metabolism, cell migration, calcium transport and cell-cell signaling all related to developmental changes. In-silico analysis identified conservation of 3’ UTR of Platr14 across species as well as unique sites complementary to about 29 DNA binding sites of the mouse genome. Gene Ontology studies identified these DNA binding sites to have roles related to developmental processes. In this dissertation, we identified lincRNA Platr14, to have a novel role in mesoderm lineage driving the cardiac and skeletal myogenic differentiation.