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

项目总结和摘要 RNA监视途径调节细胞RNA的质量及其丰度。在细胞核中，异常的， 错误折叠或有缺陷的RNA主要由包括RNA在内的3 - 5衰变机制降解 外泌体是一种多亚基蛋白复合体，催化3 - 5-RNA降解。核RNA外泌体 靶向底物由含有MTR 4解旋酶的几种蛋白质复合物介导。其中包括 核外泌体靶向（NEXT）复合物和多聚（A）外泌体靶向（PAXT）连接， RNA外泌体的上游。RNA外泌体突变体或过表达的辅因子有助于 许多人类疾病，包括癌症。尽管有几项研究阐明了人类外泌体 结构和功能，关于人类NEXT和PAXT如何参与和准备其功能的研究有限。 RNA外泌体是用于将其递送至RNA外泌体的底物。 这项研究将从事生物化学，结构和功能的方法来表征核心 PAXT复合物（目标1），并确定底物如何被识别为核糖核蛋白（RNP）复合物 通过核帽结合复合物和多聚腺苷酸结合蛋白（Aim 2）。我会利用试剂 以及在K99阶段Aim 1和Aim 2中开发的研究microRNA（miRNA）加工的技能 和通过PAXT连接的营业额（目标3）。这项研究的结果将提供新的见解， PAXT识别靶RNP的分子机制及其与RNA外泌体的相互作用 衰变途径此外，该研究将扩展到miRNA生物发生。作为RNA衰变途径， miRNA的生物合成在基因调控和癌症中起着基础性的作用，这项研究可能会影响人类的健康 NCI的使命是发展科学知识，支持所有人活得更长、更健康。 这份建议书描述了一个全面的培训计划，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 处理.这个奖项的目的是鼓励和准备我的独立研究计划，我 我将利用这段时间来提高我的科学知识和专业技能。在K99阶段，我将 采用生物化学方法重建PAXT与其靶点的连接，并将它们与联合收割机结合， 冷冻电子显微镜在Christopher D.利马的指导。这将扩展我的技术储备 以及RNA生物学、癌症生物学和结构生物学方面的生物学知识，以供未来研究之用。还有， 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.