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

Poly(A) 外泌体靶向 (PAXT) 复合物识别核糖核蛋白 (RNP) 底物

• 批准号: 10360355
• 负责人: Mi Seul Park
• 金额: $ 11.92万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10360355
• 关键词: Acute Myelocytic Leukemia; Address; Arabidopsis; Arsenites; Award; Binding; Binding Proteins; Biochemical; Biogenesis; Biological; Biological Assay; Biology; Biophysics; Cancer Biology; Cell Nucleus; Cell Proliferation; Cholangiocarcinoma; Complex; Cryoelectron Microscopy; Cytoplasm; DNA-Directed RNA Polymerase; Defect; Development; Environment; Exoribonucleases; Gene Expression; Gene Expression Regulation; Goals; Health; Homologous Gene; Human; Individual; Knowledge; Laboratory Research; Malignant Neoplasms; Mediating; Memorial Sloan-Kettering Cancer Center; Mentors; Messenger RNA; Methods; MicroRNAs; Microprocessor; Mission; Modeling; Molecular; Monitor; Mutate; Mutation; Nuclear; Nuclear RNA; Outcome Study; Output; Pathway interactions; Persons; Phase; Plants; Play; Poly A; Poly(A) Tail; Poly(A)+ RNA; Poly(A)-Binding Proteins; Polymerase; Primary carcinoma of the liver cells; Process; Protein Complex Subunit; Proteins; RNA; RNA Binding; RNA Cap-Binding Proteins; RNA Caps; RNA Decay; RNA Degradation; RNA Polymerase II; RNASE3L gene; Reagent; Regulation; Research; Resistance; Resources; Ribonucleoproteins; Role; Structure; Training; Transcript; Zinc; Zinc Fingers; career; career development; cofactor; exosome; helicase; human disease; in vivo; insight; mutant; novel; overexpression; particle; premature; programs; protein complex; reconstitution; skills; structural biology; transcriptome; translocase

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.

项目概要和摘要 RNA 监测途径调节细胞 RNA 的质量及其丰度。在细胞核中，异常， 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，一种多亚基蛋白质复合物，可催化 3 至 5 厘 RNA 降解。核RNA外泌体 靶向底物由多种含有 MTR4 解旋酶的蛋白质复合物介导。这些包括 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外泌体突变体或过度表达的辅助因子有助于 许多人类疾病，包括癌症。尽管一些研究阐明了人类外泌体 结构和功能，关于人类 NEXT 和 PAXT 如何参与和准备它们的研究有限 将其递送至 RNA 外泌体的底物。 这项研究将采用生物化学、结构和功能方法来表征核心 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 Poly(A) 结合蛋白（目标 2）。我将利用试剂 以及 K99 阶段在目标 1 和目标 2 中开发的技能，以研究 microRNA (miRNA) 加工 通过 PAXT 连接实现营业额（目标 3）。这项研究的结果将为我们提供新的见解 PAXT识别目标RNP的分子机制及其与RNA中RNA外泌体的相互作用 衰变途径。此外，该研究还将扩展到 miRNA 生物合成。随着RNA衰变途径和 miRNA 生物发生在基因调控和癌症中发挥基础作用，这项研究可以影响人类健康 NCI 的使命是发展科学知识并支持所有人活得更长久、更健康。 该提案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 衰减和初级 miRNA 加工。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，我会 采用生化方法重建 PAXT 与其靶标的连接，并将其与 在 Christopher D. Lima 博士的指导下进行冷冻电子显微镜。这将扩展我的技术能力 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，以供未来研究使用。还， 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。