PROJECT SUMMARY/ABSTRACT Precisely controlling the expression levels of genes is essential to any biological process. microRNAs (miRNAs) play important roles in controlling gene expression by binding to specific target mRNAs and downregulating their expression. To understand how a miRNA functions it is critical to both identify the set of mRNA targets to which it binds in a specific biological context, and to determine how this pairing interaction regulates gene expression to ultimately impact cellular processes and phenotypes. Some miRNAs can be controlled by competing endogenous RNAs (ceRNAs), which bind miRNAs and prevent them from downregulating expression of their mRNA targets, creating a complex network of regulation that can be difficult to experimentally unravel. The proposed research will develop a comprehensive experimental approach to uncover the biological functions of a miRNA. As a model system the work focuses on two miRNAs (miR-1 and miR-206) that control myogenesis, the differentiation of skeletal muscle cells. The experiments use cultured mouse C2C12 cells, a widely used model for myogenesis, as well as in vivo experiments with mice. In Specific Aim 1 experiments will identify the direct RNA targets of miR-1 and miR-206 in undifferentiated C2C12 cells and during differentiation. Supporting data demonstrate the success of a target identification technique, which will be used with miR-1 and miR-206 knockout cells to identify mRNAs uniquely targeted by each miRNA. In Specific Aim 2, newly identified targets of miR-1 and miR-206 during myogenesis will be screened for their contributions to differentiation phenotypes. Experiments will also test the function of miRNA/target pairing in controlling the expression levels of targets during myogenesis. In addition, the regulatory relationships of specific miRNA/ target pairs will be studied using mouse models. In Specific Aim 3 experiments will test the hypothesis that RNA targets of miR-1 and miR-206 in undifferentiated cells function as ceRNAs to maintain proliferation and prevent premature differentiation. The absolute levels of miRNAs and their targets in undifferentiated cells will be quantified, targets will be screened to identify those important for maintaining cellular proliferation, and regulatory mechanisms of ceRNAs will be investigated. Together the proposed studies develop an experimental framework to understand the functional roles of two important miRNAs. This work will not only advance understanding of the roles of miR-1 and miR-206 in myogenesis, but will also provide a comprehensive strategy that could be applied to other miRNAs in a variety of biological systems.