Recognition of ribonucleoprotein (RNP) substrate by the poly(A) exosome targeting (PAXT) complex

项目摘要和摘要 RNA监视途径调节细胞RNA的质量和丰度。在细胞核中，异常的， 错误折叠或有缺陷的RNA主要通过包括RNA在内的3？-5？衰变机制降解 外切体，是一种多亚单位的蛋白质复合体，催化3？5？RNA的降解。核糖核糖核酸外切体 靶向底物是由几个含有MTR4解旋酶的蛋白质复合体介导的。其中包括 核外切体靶向(NEXT)复合体及其与聚(A)外切体靶向(PAXT)的连接 位于RNA外切体的上游。RNA外切体突变体或过度表达的辅因子有助于 无数的人类疾病，包括癌症。尽管有几项研究阐明了人类外切体 结构和功能，关于人类Next和PAXT如何参与和准备它们的研究有限 将它们输送到RNA外切体的底物。 这项研究将采用生化、结构和功能方法来表征核心 PAXT复合体(目标1)，并确定底物如何被识别为核糖核蛋白(RNP)复合体 通过核帽结合复合体和聚(A)结合蛋白(目标2)。我会利用试剂的使用 以及在K99阶段在目标1和目标2中发展的技能，以研究microRNA(MiRNA)的加工 和通过PAXT连接的营业额(目标3)。这项研究的结果将为研究 PAXT识别靶RNP的分子机制及其与RNA外切体的相互作用 腐烂的路径。此外，这项研究将扩展到miRNA生物发生。随着RNA衰变途径和 MiRNA的生物发生在基因调控和癌症中起着基础性作用，这项研究可能会影响人类健康 NCI的使命是发展科学知识，支持所有人活得更长、更健康。 这份建议描述了一项全面的培训计划，以通过调查来发展我的学术生涯 PAXT如何识别其RNP底物以刺激下游RNA衰变和初级miRNA 正在处理。这个奖项的目的是鼓励和准备我的独立研究项目，我 我会利用这段时间提升我的科学知识和专业技能。在K99阶段，我将 使用生化方法重建PAXT与其靶标的连接并将其与 克里斯托弗·D·利马博士指导下的低温电子显微镜。这将扩展我的技术曲目 以及RNA生物学、癌症生物学和结构生物学的生物学知识，为未来的研究做准备。另外， 纪念斯隆·凯特琳癌症中心将提供机构支持，包括实验室研究 资源，开放的科学环境，和职业发展来实现我的目标。

PROJECT SUMMARY AND ABSTRACT RNA surveillance pathways regulate the quality of cellular RNAs and their abundance. In the nucleus, aberrant, misfolded, or defective RNAs are degraded primarily by the 3¢-5¢ decay machinery that includes the RNA exosome, a multi-subunit protein complex that catalyzes 3¢ to 5¢ RNA degradation. The nuclear RNA exosome targeting substrate is mediated by several protein complexes containing the MTR4 helicase. These include the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection which lie upstream of the RNA exosome. The RNA exosome mutants or overexpressed cofactors contribute to numerous human diseases, including cancer. Although several studies illuminated the human exosome structure and functions, there are limited studies on how human NEXT and PAXT engage and prepare their substrates for their delivery to the RNA exosome. This research will engage biochemical, structural, and functional approaches to characterize the core PAXT complex (Aim 1) and determine how substrates are recognized as ribonucleoprotein (RNP) complexes through nuclear cap-binding complex and poly(A) binding protein (Aim 2). I will leverage the use of reagents and skills developed in Aim 1 and Aim 2 during the K99 phase to investigate microRNA (miRNA) processing and turnover by PAXT connection (Aim 3). The outcome of this study will provide novel insights into the molecular mechanism of target RNP recognition by PAXT and their interaction with the RNA exosome in RNA decay pathways. Moreover, the study will be expanding to miRNA biogenesis. As RNA decay pathways and miRNA biogenesis play a fundamental role in gene regulation and cancer, this study can impact human health and the NCI mission to develop scientific knowledge and support all people to live longer and healthier. This proposal describes a comprehensive training plan to develop my academic career by investigating how PAXT recognizes its RNP substrates to stimulate downstream RNA decay and primary miRNA processing. The purpose of this award is to encourage and prepare my independent research program, and I will use this period to advance my scientific knowledge and professional skills. During the K99 phase, I will employ biochemical approaches to reconstitute the PAXT connection with its target and combine them with cryo-electron microscopy under Dr. Christopher D. Lima’s mentoring. That will extend my technical repertoire and biological knowledge in RNA biology, cancer biology, and structural biology for future research. Also, Memorial Sloan Kettering Cancer Center will provide institutional support, including laboratory research resources, an open scientific environment, and career development to achieve my goals.