STRUCTURES OF EXOSOME AND SIGNAL RECOGNITION PARTICLE

外泌体和信号识别颗粒的结构

• 批准号: 7955546
• 负责人: Ailong Ke
• 金额: $ 5.23万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955546
• 关键词: 3' Untranslated Regions; 5' -exoribonuclease; Adaptor Signaling Protein; Architecture; Cell Nucleus; Cell Survival; Cell physiology; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Cytoplasm; Defect; Elements; Enzymatic Biochemistry; Eukaryota; Eukaryotic Cell; Funding; Goals; Grant; Guanosine Triphosphate; Hand; Hydrolysis; Institution; Life; Mediating; Membrane; Mutation; Nomenclature; Nonsense Codon; Nuclear; Peptide Signal Sequences; Phase; Process; Protein translocation; Proteins; RNA; RNA Processing; RNA Splicing; Regulation; Research; Research Personnel; Resolution; Resources; Ribonucleoproteins; Ribosomal RNA; Ribosomes; Roentgen Rays; Signal Recognition Particle; Source; Structure; Terminator Codon; Time; Translating; Translations; United States National Institutes of Health; Yeasts; insight; multicatalytic endopeptidase complex; protein complex; signal recognition particle receptor; synchrotron radiation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Part of my research focuses on a crucial RNA processing and degradation machinery, the exosome, which has often been called the proteasome for RNA. Initially identified from mutations causing 5.8S rRNA 3' end processing defects, the exosome is a conserved 300 - 400 kD 3'-to-5' exoribonuclease complex present in both the nucleus and the cytoplasm of eukaryotic cells. The exosome consists of a core of ten proteins: Rrp4p, Rrp40p to Rrp46p, Mtr3p, and Csl4p (yeast nomenclature), all are essential for cell viability. Six of them are phosphorolytic RNases; the other four are predicted to be hydrolytic RNases. In the nucleus, the exosome is required for the 3' end formation of 5.8S rRNA and the degradation of the 5'-external transcribed spacer; participated in the 3' end maturation of small nuclear and nucleolar RNAs; and involved in degradation of inefficiently spliced or hypoadenylated pre-mRNAs. The cytoplasmic exosome is involved in the degradation of mRNAs containing premature termination codons, lacking termination codons, or bearing AU-rich elements (AREs) near the 3' untranslated region. To gain insights into the architecture and enzymatic mechanism of the exosome, I have proposed to determine the crystal structures of the 300 kD, 4-preotein archaeal exosome, the 10-protein eukaryotic exosome, and the exosome-RNA substrate complexes. The long term goals include determination of exosome-adaptor protein complexes to investigate its regulation. Exciting progress has been made in expression, purification, and crystallization of the archaeal exosome complex. Well-behaving crystals diffracted X-ray to ~ 2.4 ¿ resolution at synchrotron radiation source. Additional beam time is needed to complete Se-MAD phasing and carry out structural enzymology studies on the archaeal exosome complex. The second part of my research focuses on Signal Recognition Particle (SRP)  mediated co-translational translocation of proteins across or into membranes. This vital cellular process requires the translating ribosome to be membrane-targeted by the SRP, a ribonucleoprotein complex conserved in all three kingdoms of life. SRP recognizes the hydrophobic signal sequence of the nascent protein emerging from the ribosome, resulting in transient elongation arrest in eukaryotes, and targets the ribosome to the membrane via a GTP-dependent interaction with the SRP receptor (SR). The ribosome is then handed over to the translocon, where protein translation and translocation happens simultaneously. The SRP-SR dissociates following GTP hydrolysis and SRP cycle resumes.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 我的部分研究重点是关键的 RNA 加工和降解机制，即外泌体，通常被称为 RNA 蛋白酶体，最初是从导致 5.8S rRNA 3' 末端加工缺陷的突变中发现的，外泌体是一种保守的 300 - 400 kD。 3'-to-5'外核糖核酸酶复合物存在于真核细胞的细胞核和细胞质中，外泌体由十种蛋白质的核心组成：Rrp4p、 Rrp40p、Rrp46p、Mtr3p 和 Csl4p（酵母命名法）均对细胞活力至关重要，其中 6 种是磷酸解 RNase；另外 4 种预计是细胞核中的水解 RNase，外泌体是 3' 末端所必需的。 5.8S rRNA 的形成和 5'-外部转录间隔区的降解参与了 3' 末端的成熟；细胞核和核仁 RNA 的降解；并参与低效剪接或腺苷酸化前体 mRNA 的降解 细胞质外泌体参与含有过早终止密码子、缺乏终止密码子或富含 AU 元件 (ARE) 的 mRNA 的降解。靠近3'非翻译区。 为了深入了解外泌体的结构和酶促机制，我建议确定 300 kD、4-蛋白质古菌外泌体、10 蛋白真核外泌体和外泌体-RNA 底物复合物的晶体结构。目标包括确定外泌体-接头蛋白复合物以研究其调节作用，在古细菌外泌体复合物的表达、纯化和结晶方面取得了令人兴奋的进展。表现良好的晶体 X 射线衍射至 ~ 2.4 ¿需要额外的光束时间来完成 Se-MAD 定相并对古菌外泌体复合物进行结构酶学研究。 我研究的第二部分重点是信号识别颗粒 (SRP) 介导的蛋白质跨膜或跨膜共翻译易位，这一重要的细胞过程需要 SRP（一种在所有三种细胞中都保守的核糖核蛋白复合物）对翻译核糖体进行膜靶向。 SRP 识别核糖体中出现的新生蛋白的疏水信号序列，导致真核生物中短暂的伸长停滞，并靶向核糖体。然后，核糖体通过与 SRP 受体 (SR) 的 GTP 依赖性相互作用转移到膜上，其中蛋白质翻译和易位在 GTP 水解和 SRP 循环恢复后同时发生。