Identify the Transcriptome and Proteome Associated with miRNAs dring Myogenesis

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 DESCRIPTION (provided by applicant): MicroRNAs play a fundamental role in post-transcriptional control of gene expression. They are key participants in the differentiation of skeletal muscle cells and are implicated in many different skeletal muscle diseases. It is of fundamental importance; therefore, to identify the direct, bona fide mRNA targets of individual microRNAs, as well as the proteins that each associates with throughout its lifecycle in differentiating muscle cells. Doing so will reveal the biological underpinnings for why select microRNAs are themselves dysregulated in disease states as well as why the resulting aberrant regulation of their mRNA targets contribute to disease progression. The broad objective of the proposed research is to develop methods to trap and purify microRNA-mRNA-protein complexes and identify the mRNAs and proteins associated with specific microRNAs. These methods will directly connect a microRNA with its target transcripts, as well as determine the proteins associated with the microRNA throughout its lifecycle. Central to the method development is affinity purification using antisense oligos to isolate individual endogenous microRNAs and their associated biomolecules from crosslinked cells, referred to as xOP (crosslinking oligo purification). The aims of the proposal are: Aim 1. Develop a technique to experimentally define the target transcriptome of individual miRNAs important in myogenesis. Using two model microRNAs upregulated in differentiating muscle cells, an approach will be developed to rapidly and accurately define the direct mRNA targets of any given microRNA by coupling xOP purification to deep sequencing (xOP-seq). These experiments will reveal the transcriptomes associated with two endogenous microRNAs in differentiating myoblasts, thereby providing significant insight into their role in myogenesis. Aim 2. Develop a technique to identify the interaction proteome of individual miRNAs important in myogenesis. A two-pronged approach will be developed that will identify the proteins that are directly and indirectly associated with two different microRNAs in differentiating muscle cells by coupling xOP purification to mass spectrometry. The xOP-MS technique will reveal the complete interaction proteome of two microRNAs as they are post-transcriptionally processed and ultimately used to control gene expression during myogenesis. Both techniques that will be developed have the potential to transform the study of microRNA processing and function, as well as provide significant insight into the role of two species microRNAs during muscle cell differentiation. Currently, there is no method to experimentally identify the set of direct mRNA targets of an endogenous microRNA, nor is there a method to identify the set of proteins that associate with an endogenous microRNA throughout its cellular lifecycle. Developing methods to reliably and rapidly obtain this information will provide researchers with technology to gain unprecedented insight into the roles that many microRNAs play in skeletal muscle development and disease.