PROJECT SUMMARY/ABSTRACT Post-transcriptional control permeates biology from proliferation to development. RNA-binding proteins (RBPs) dictate which messenger (m)RNAs are regulated, and how, where, and when that regulation occurs. Their roles in biology are incontrovertible, and emphasized by RBP dysfunctions that cause disease, including cancer, obesity and muscular atrophies. The long-term goal is to understand how RNA-protein complexes dictate and respond to complex biological events to realize how defects in these complexes result in disease. The objective of this proposal is to understand the diverse post-transcriptional regulation of the RBP YBX3, and connect its regulation to key biological processes and disease. The central hypothesis, which was formulated based on previous findings and preliminary data, is that YBX3 post-transcriptionally regulates mRNAs via multiple mechanisms, and this control is required to maintain amino acid transport in skeletal muscle. A multi-disciplinary approach that combines biochemistry, “omics”, bioinformatics and mammalian cell culture to dissect how YBX3 regulates via diverse mechanisms, and the role for its post-transcriptional control of amino acid transporters in skeletal muscles. The rationale for the proposed work is that once the diverse post-transcriptional control mechanisms are understood, this can be used as a paradigm for other clinically relevant RBPs, and to potentially develop therapeutic strategies based on this regulation. The objective of this project will be accomplished by three specific aims: 1) Define how the modular domains of YBX3 contribute to post-transcriptional regulation. The working hypothesis is that the modular domains of YBX3 help determine the diverse regulatory outcomes. The investigators will modify a well-established biochemical assay to assay the regulatory contribution of each domain. 2) Identify YBX3-dependent complexes formed on specific mRNAs. The working hypothesis is that different complexes form on mRNAs that YBX3 either activates or represses. RNA pull-down approaches will be used to identify transcript specific complexes using targeted and non-targeted methods. 3) Characterize the role of YBX3's post-transcriptional control of amino acid transport in skeletal muscle. The working hypothesis is that YBX3 stabilizes transporter mRNAs to maintain amino acid homeostasis in skeletal muscle that is critical for differentiation in this tissue. Amino acid mRNA stability, the intracellular levels of amino acids and differentiation will be assessed when YBX3 is depleted in skeletal muscle cells. This proposal is innovative because it 1) establishes how a single RBP controls mRNAs via multiple mechanisms, which can be used as a paradigm for other multi-regulatory RBPs, and 2) defines how post-transcriptional regulation impacts amino acid transport in skeletal muscle that could lead to new therapeutic strategies of a process that is impaired with aging. The proposed work is significant because it will 1) provide a model of how discrete domains regulate, 2) identify mRNA specific complexes required for regulation and 3) uncover how post-transcriptional control regulates amino acid transport and differentiation in skeletal muscle.

PROJECT NARRATIVE All biological processes rely on precise control of the genetic material, and the control of mRNAs by RNA-binding proteins (RBPs) is vital for this regulation. RBP dysfunctions cause disease including cancer, epilepsy and obesity, and the richness of the problem lies in the staggeringly large number of ways RBPs exert their control, which can vary from RNA to RNA, cell to cell, and in health versus disease. This application will address these questions using a range of approaches, from biochemistry to genomics and mass spectrometry.