Mechanism of action of an lncRNA for directing muscle differentiation

 描述(申请人提供)：长非编码RNA(LncRNAs)是非编码的长(100个碱基)单链RNA，作为基因表达的主要调节因子而突然出现。MicroRNAs是大约十年前出现的大约22个碱基长的单链RNA，这个实验室是证明几个microRNAs在骨骼肌成肌细胞分化为肌管中的作用的先驱之一。我们现在建议利用我们在RNA在肌肉分化中的作用方面的专业知识来确定一种特定的肌肉分化诱导(MDI)lncRNA，MUNC，通过其调节基因表达和促进肌肉分化的机制。目的1验证MUNC对肌肉分化是必不可少的这一假设，因为它作为参与基因调控的新蛋白复合体的组成部分，调节关键的生肌转录因子的结合或表达。目的2对小鼠和人的MUNC进行结构功能研究，以验证该lncRNA中存在进化保守结构域的假设，这些结构域对其作用机制至关重要。目的3将寻找新的MUNC蛋白和DNA合作伙伴，以确定MUNC的独立于肌源性转录因子的功能。这一目标还将识别在肌肉分化过程中诱导的其他lncRNAs，并将它们在分化中的作用与MUNC和MyoD结合起来。这是一个令人兴奋的项目，涉及一名分子生物学家和一名骨骼肌生理学家的合作。它将使用新的实验试剂，如lncRNA和最先进的方法，如chirp。LC-MS-MS、SILAC和Chirp-seq，以发现一个lncRNA在控制细胞类型特异性方面的作用机制。由于我们的初步结果发现了在分化过程中诱导的数十个新的lncRNAs，其中至少有一些已经与分化有关，该项目的成功将启动对这一非常重要的分化范式中的几个lncRNAs的长期研究。

 DESCRIPTION (provided by applicant): Long noncoding RNAs (lncRNAs) are noncoding long (>100 bases) single- stranded RNAs that have burst onto the scene as major regulators of gene expression. MicroRNAs, are about 22 base long single-stranded RNAs that came to the fore about a decade back and this laboratory has been among the pioneers that demonstrated the roles of several microRNAs in the differentiation of skeletal muscle myoblasts to myotubes. We now propose to use our expertise in the role of RNAs in muscle differentiation to determine the mechanism by which one particular muscle- differentiation-induced (MDI) lncRNA, MUNC, regulates gene expression and promotes muscle differentiation. Aim 1 will test the hypothesis that MUNC is essential for muscle differentiation because of its role in either regulating the binding or the expression of critical myogenic transcription factors as a constituent of novel protein complexes involved in gene regulation. Aim 2 will carry out structure function studies on mouse and human MUNC to test the hypothesis that evolutionarily conserved domains exist in this lncRNA that are critical for its mechanism of action. Aim 3 will identify new protein and DNA partners of MUNC to identify functions of MUNC independent of Myogenic transcription factors. This Aim will also identify additional lncRNAs that are induced during muscle differentiation and integrate their role in differentiation with that of MUNC and MyoD. This is an exciting project that involves the collaboration of a molecular biologist with a skeletal muscle physiologist. It wil use new experimental reagents such as lncRNAs and state-of-the-art approaches like ChIRP. LC-MS-MS, SILAC and ChiRP-seq to discover the mechanism of action of one lncRNA in control of cell-type specificity. Since our preliminary results identify tens of novel lncRNAs induced during differentiation, at least some of which already implicated in differentiation, success of this project will launch a prolonged investigation of several lncRNAs in this very important paradigm of differentiation.