Post-transcriptional regulation by the YBX3 RNA-binding protein in skeletal muscle

项目总结/摘要 转录后调控贯穿于从增殖到发育的生物学过程。RNA结合蛋白 决定哪些信使（m）RNA受到调控，以及调控如何、在何处和何时发生。他们的角色 在生物学上是无可争议的，并强调由RBP功能障碍，导致疾病，包括癌症， 肥胖和肌肉萎缩。长期目标是了解RNA-蛋白质复合物是如何决定和 对复杂的生物事件作出反应，以了解这些复合物中的缺陷如何导致疾病。客观 这项建议的目的是了解RBP YBX 3的多种转录后调控，并将其 调节关键的生物过程和疾病。中心假设是根据以下内容制定的 先前的发现和初步数据表明，YBX 3通过多种途径转录后调节mRNA， 这种控制是维持骨骼肌中氨基酸转运所必需的。一个多学科 一种结合生物化学、“组学”、生物信息学和哺乳动物细胞培养的方法， 调节通过不同的机制，其作用的转录后控制氨基酸转运蛋白， 骨骼肌这项工作的基本原理是，一旦不同的转录后控制 了解机制，这可以用作其他临床相关RBP的范例，并可能 根据这一规定制定治疗策略。本项目的目标将通过以下方式实现： 三个具体目标：1）定义YBX 3的模块结构域如何有助于转录后调节。 工作假设是，YBX 3的模块化结构域有助于确定不同的监管结果。 研究人员将修改一个完善的生化测定，以测定每一个基因的调节作用。 域2)识别特定mRNA上形成的YBX 3依赖性复合物。工作假设是， YBX 3激活或抑制的mRNA上形成不同的复合物。RNA下拉方法将是 用于使用靶向和非靶向方法鉴定转录物特异性复合物。3)描述角色 YBX 3在骨骼肌中对氨基酸转运的转录后调控。工作假设是， YBX 3稳定转运蛋白mRNA以维持骨骼肌中的氨基酸稳态，这对于 在这个组织中的分化。氨基酸mRNA的稳定性、细胞内氨基酸水平和分化 将在骨骼肌细胞中耗尽YBX 3时进行评估。这是一项创新，因为它（1） 建立了一个单一的RBP如何通过多种机制控制mRNA，这可以作为一个范例， 其他多调节RBP，和2）定义了转录后调节如何影响氨基酸转运， 骨骼肌，可能导致新的治疗策略的过程中，是受损的老化。的 拟议的工作是重要的，因为它将1）提供一个模型，如何离散域调节，2）确定 mRNA特异性复合物的调控和3）揭示如何转录后控制调节 氨基酸转运和骨骼肌分化。

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.