Identify the Transcriptome and Proteome Associated with miRNAs dring Myogenesis

鉴定与肌发生过程中 miRNA 相关的转录组和蛋白质组

• 批准号: 8866691
• 负责人: Jennifer F. Kugel
• 金额: $ 16.87万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8866691
• 关键词: Affinity Chromatography; Applications Grants; Biological; Biological Models; Cell Differentiation process; Cells; Complex; Computing Methodologies; Coupled; Coupling; Detection; Development; Disease; Disease Progression; Ectopic Expression; Gene Expression; Gene Expression Profile; Genes; Goals; Growth and Development function; Individual; Knowledge; Lead; Life; Mass Spectrum Analysis; Messenger RNA; Methods; MicroRNAs; Modeling; Muscle Cells; Muscle Development; Muscle Fibers; Myoblasts; Myopathy; Normal Cell; Normal tissue morphology; Oligonucleotides; Outcome; Participant; Play; Positioning Attribute; Post-Transcriptional Regulation; Process; Protein Binding; Proteins; Proteome; RNA-Protein Interaction; Regulation; Research; Research Personnel; Risk; Role; Set protein; Skeletal Muscle; Techniques; Technology; Tissues; Transcript; cell growth; cell type; crosslink; deep sequencing; high risk; insight; method development; myogenesis; new technology; protein complex; public health relevance; rapid technique; research study; skeletal muscle differentiation

 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.

 描述（由申请人提供）：MicroRNA在基因表达的转录后控制中发挥重要作用。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，识别单个microRNA的直接、真正的mRNA靶点以及在分化肌细胞的整个生命周期中与每个microRNA相关的蛋白质是至关重要的。这样做将揭示为什么选择microRNA本身在疾病状态下失调的生物学基础，以及为什么导致其mRNA靶点的异常调节有助于疾病进展。该研究的主要目标是开发捕获和纯化microRNA-mRNA-蛋白质复合物的方法，并鉴定与特定microRNA相关的mRNA和蛋白质。这些方法将直接将microRNA与其靶转录物连接起来，并确定与microRNA整个生命周期相关的蛋白质。方法开发的核心是使用反义寡核苷酸进行亲和纯化，以从交联细胞中分离单个内源性microRNA及其相关生物分子，称为xOP（交联寡核苷酸纯化）。 该提案的目标是：目标1。开发一种技术来实验性地确定在肌生成中重要的单个miRNA的靶转录组。使用在分化的肌肉细胞中上调的两种模型microRNA，将开发一种方法，通过将xOP纯化与深度测序（xOP-seq）结合来快速准确地确定任何给定microRNA的直接mRNA靶标。这些实验将揭示与分化成肌细胞中的两种内源性microRNA相关的转录组，从而为它们在肌发生中的作用提供重要的见解。目标二。开发一种技术，以确定在肌肉发生中重要的单个miRNA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纯化与质谱法结合，鉴定与两种不同microRNA直接和间接相关的蛋白质，以分化肌细胞。xOP-MS技术将揭示两种microRNA的完整相互作用蛋白质组，因为它们是转录后加工的，并最终用于控制肌发生过程中的基因表达。 这两种技术都有可能改变microRNA加工和功能的研究，并为两种microRNA在肌肉细胞分化过程中的作用提供重要的见解。目前，还没有方法通过实验鉴定内源性微小RNA的一组直接mRNA靶点，也没有方法鉴定在整个细胞生命周期中与内源性微小RNA相关的一组蛋白质。开发可靠和快速获得这些信息的方法将为研究人员提供技术，以获得对许多microRNA在骨骼肌发育和疾病中所起作用的前所未有的洞察力。