Biogenesis and Function of Guide RNAs

向导RNA的生物发生和功能

基本信息

批准号：
8509113
负责人：
Ruslan Afasizhev
金额：
$ 34.22万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2013-05-16
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8509113
关键词：
Affinity African Trypanosomiasis American Animal Model Area Binding Biogenesis Biological Process Cell Line Code Complex Core Protein DNA Structure Data Developing Countries Diphosphates Event Gene Expression Generations Genetic Genome Guide RNA Health Holoenzymes Hybrids Hydrolase Hydrolysis In Vitro Maintenance Mediating Messenger RNA Metabolic Methods Mitochondria Molecular Molecular Biology Nuclear Parasites Parasitic Diseases Pathway interactions Pattern Pharmaceutical Preparations Phosphorylation Polyadenylation Process Protein Overexpression Proteins Proteomics Protozoa RNA RNA Binding RNA Editing RNA Helicase RNA Interference RNA Precursors RNA Processing RNA Stability RNA annealing RNA-Binding Proteins Reaction Recycling Relative (related person)Ribonucleoproteins Ribosomal RNA Specific qualifier value Structure Tail Testing Transcript Translations Trypanocidal Agents Trypanosoma Trypanosoma brucei brucei base insertion/deletion mutation molecular mass overexpression particle pathogen polypeptide prevent protein complex reconstitution therapeutic target tripolyphosphate

项目摘要

DESCRIPTION (provided by applicant): Trypanosomes are parasitic protozoa responsible for health problems in developing countries, including those with a substantial U.S. presence. Along with being an important pathogen, Trypanosoma brucei offers an expanded molecular biology landscape that extends beyond that of the standard set of model organisms. Indeed, many unique biological processes have been discovered in this parasite. In particular, its giant mitochondrion encloses an unusual DNA structure, referred to as the kinetoplast, and unconventional RNA processing pathways. The kinetoplast carries a protein-coding maxicircle genome catenated to minicircles which encode a vast array of guide RNAs (gRNAs). A multitude of nuclear-encoded factors mediate the interactions between maxi- and minicircle transcripts to create functional mRNAs via nucleolytic processing, U- insertion/deletion RNA editing, and 32 polyadenylation. Editing reactions are catalyzed by the RNA editing core complex, RECC (20S editosome), while each step of the enzymatic cascade is directed by gRNAs. Although studies of RECC have shown impressive progress, until recently little was known about gRNA biogenesis, stabilization, recruitment to the editing process, and post-editing metabolic fate. We discovered that RNA editing TUTase 1 (RET1) is involved in the nucleolytic processing of gRNA precursors and that mature gRNAs are stabilized via binding to gRNA binding complex subunits 1 and 2 (GRBC1/2). In preliminary studies we identified GRBC1/2-associated proteins, including NUDIX hydrolases, DExD/H RNA helicases, RNA binding proteins, and polypeptides lacking identifiable domains. The overall complexity of GRBC likely exceeds that of the RECC, which consists of ~20 proteins. This proposal focuses on GRBC protein composition, mechanisms of gRNA binding and commitment to the editing process, and post-editing gRNA displacement. We hypothesize that interacting RECC and GRBC form an RNA editing holoenzyme, termed the 40S editosome, and propose to: 1) define core protein components and those involved in RNA-mediated contacts within GRBC; 2) determine the mechanisms of gRNA binding to GRBC1/2 as well as GRBC-RECC interaction; and 3) investigate the molecular basis of post-editing gRNA displacement.

描述（由申请人提供）：锥虫是一种寄生原生动物，会导致发展中国家（包括在美国大量存在的国家）的健康问题。除了作为一种重要的病原体之外，布氏锥虫还提供了扩展的分子生物学景观，超出了标准模式生物的范围。事实上，在这种寄生虫中已经发现了许多独特的生物过程。特别是，其巨大的线粒体包围着一种不寻常的 DNA 结构（称为动质体）和非常规的 RNA 加工途径。动质体携带一个编码蛋白质的大环基因组，该基因组与编码大量引导 RNA (gRNA) 的小环相连。多种核编码因子介导大环和小环转录物之间的相互作用，通过溶核处理、U-插入/删除 RNA 编辑和 32 聚腺苷酸化来创建功能性 mRNA。编辑反应由 RNA 编辑核心复合物 RECC（20S 编辑体）催化，而酶级联的每一步均由 gRNA 指导。尽管 RECC 研究取得了令人瞩目的进展，但直到最近，人们对 gRNA 的生物发生、稳定性、编辑过程的招募以及编辑后代谢命运知之甚少。我们发现 RNA 编辑 TUTase 1 (RET1) 参与 gRNA 前体的溶核加工，并且成熟的 gRNA 通过与 gRNA 结合复合物亚基 1 和 2 (GRBC1/2) 结合而稳定。在初步研究中，我们鉴定了 GRBC1/2 相关蛋白，包括 NUDIX 水解酶、DExD/H RNA 解旋酶、RNA 结合蛋白和缺乏可识别结构域的多肽。 GRBC 的整体复杂性可能超过 RECC，后者由约 20 个蛋白质组成。该提案重点关注 GRBC 蛋白组成、gRNA 结合机制和对编辑过程的承诺，以及编辑后 gRNA 置换。我们假设 RECC 和 GRBC 相互作用形成一种 RNA 编辑全酶，称为 40S 编辑体，并提出：1）定义核心蛋白成分和那些参与 GRBC 内 RNA 介导的接触的蛋白成分； 2）确定gRNA与GRBC1/2结合以及GRBC-RECC相互作用的机制； 3) 研究编辑后 gRNA 置换的分子基础。