Identify the Transcriptome and Proteome Associated with miRNAs dring Myogenesis

 描述(由申请人提供)：microRNAs在基因表达的转录后控制中起着重要作用。它们是骨骼肌细胞分化的关键参与者，并与许多不同的骨骼肌疾病有关。因此，确定单个microRNAs的直接、真诚的mRNA靶标以及每个microRNAs在分化肌肉细胞的整个生命周期中与之相关的蛋白质是至关重要的。这样做将揭示为什么选定的microRNAs本身在疾病状态下调控失调的生物学基础，以及为什么由此导致的对其mRNA靶标的异常调控有助于疾病的进展。这项研究的主要目标是开发捕获和纯化microRNA-mRNA-蛋白质复合体的方法，并鉴定与特定microRNA相关的mRNAs和蛋白质。这些方法将直接将microRNA与其目标转录本连接，并在其整个生命周期中确定与microRNA相关的蛋白质。该方法开发的核心是使用反义寡核苷酸从交联细胞中分离单个内源microRNA及其相关生物分子的亲和力纯化，称为xOP(交联寡核苷酸纯化)。该提案的目的是：目的1.开发一种技术来实验确定在肌肉发生中重要的单个miRNAs的目标转录组。利用两个在肌肉细胞分化中上调的模型microRNA，将开发一种方法，通过将xOP纯化与深度测序(xOP-seq)相结合，快速而准确地确定任何给定microRNA的直接mRNA靶标。这些实验将揭示与两个内源性microRNAs相关的转录本在分化成肌细胞中的作用，从而为了解它们在肌肉发生中的作用提供重要的见解。目的2.发展一种技术来鉴定在肌肉发生中重要的单个miRNAs的相互作用蛋白质组。将开发一种双管齐下的方法，通过将xOP纯化与质谱学相结合，在分化肌肉细胞的过程中识别与两个不同microRNAs直接和间接相关的蛋白质。XOP-MS技术将揭示两个microRNAs的完整相互作用蛋白质组，因为它们被转录后处理，并最终用于控制肌肉发生过程中的基因表达。这两项即将开发的技术都有可能改变对microRNA加工和功能的研究，并对两种microRNA在肌肉细胞分化中的作用提供重要的见解。目前，还没有方法在实验上识别内源性microRNA的一组直接mRNA靶标，也没有方法识别与内源性microRNA在细胞生命周期中相关的一组蛋白质。开发可靠而快速地获取这些信息的方法将为研究人员提供技术，以前所未有地深入了解许多microRNAs在骨骼肌发育和疾病中所扮演的角色。

 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.