STRUCTURES OF EXOSOME AND SIGNAL RECOGNITION PARTICLE

外泌体和信号识别颗粒的结构

• 批准号: 8171490
• 负责人: Ailong Ke
• 金额: $ 4.66万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171490
• 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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 我的研究的一部分着重于至关重要的RNA加工和降解机制，即外来体，通常称为RNA的蛋白酶体。最初是从导致5.8s rRNA 3末端加工缺陷的突变中鉴定出来的，外壳是一种保守的300-400 kd 3'to-5'散核酸酶复合酶，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至RRP46P，MTR3P和CSL4P（酵母命名），这对于细胞活力都是必不可少的。其中六个是磷酸化RNass。预计其他四个是水解RNass。在细胞核中，3'末端形成5.8s rRNA和5'-外部转录间隔物的降解所必需的外泌体；参加了小核和核RNA的3'结束；并参与降解无效的剪接或低腺苷酸化的前MRNA。细胞质外泌体参与了含有过早终止密码子，缺少终止密码子或附近3'未翻译区域附近的Au富元素（ARE）的mRNA降解。 为了洞悉外部体的结构和酶促机制，我提议确定300 kD，4-荧光蛋白古细胞外泌体，10蛋白质真核外泌体和外泌体-RNA底物复合物的晶体结构。长期目标包括确定外泌体适应器蛋白复合物以研究其调节。令人兴奋的进步在古细胞外泌体配合物的表达，纯化和结晶方面取得了进步。良好的晶体衍射X射线至〜2.4»同步辐射源的分辨率。需要额外的光束时间来完成SE-MAD相位，并就古细胞外泌体复合体进行结构性酶学研究。 我的研究的第二部分重点是信号识别粒子（SRP）介导的蛋白质跨或进入膜的共转移。这种重要的细胞过程要求将翻译核糖体由SRP靶向膜，这是一种在生命的所有三个王国中保守的核糖核蛋白络合物。 SRP识别从核糖体出现的新生蛋白的疏水信号序列，从而导致真核生物中的短暂伸长停滞，并通过与SRP受体（SR）的GTP相互作用将核糖体靶向膜到膜。然后将核糖体移交给Clressocon，在那里蛋白质翻译和易位的发生很简单。 GTP水解和SRP循环恢复后SRP-SR解离。