RNA Conformation and Catalysis

RNA构象和催化

• 批准号: 8403006
• 负责人: DAVID P BARTEL
• 金额: $ 37.64万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8403006
• 关键词: Animals; Binding; Biochemical; Biological Process; Biology; Candida albicans; Catalysis; Catalytic RNA; Cell physiology; Cells; Cleaved cell; Co-Immunoprecipitations; Complex; DNA; DNA-Directed RNA Polymerase; Diagnostics Research; Double-Stranded RNA; Elements; Eukaryota; Event; Evolution; Gene Expression Regulation; Gene Silencing Pathway; Genes; Genomics; Goals; Grant; Growth; Health; Heterochromatin; Human; Knock-out; Knowledge; Learning; Length; Light; Mass Spectrum Analysis; Messenger RNA; Methods; Molecular Biology; Molecular Conformation; Molecular Evolution; Monitor; Organism; Parasites; Pathway interactions; Plants; Play; Process; Production; Progress Reports; Proteins; RNA; RNA Conformation; RNA Interference; RNA Interference Pathway; RNA Polymerase II; RNA Processing; RNA Sequences; Reaction; Reagent; Regulatory Pathway; Relative (related person); Repression; Research Personnel; Retrotransposon; Ribonucleoproteins; Role; Saccharomyces; Saccharomyces cerevisiae; Saccharomycetales; Small Interfering RNA; Small RNA; Spliced Genes; Structure; Tertiary Protein Structure; Testing; Time; Translations; Viral; Virus; Work; Yeasts; chemical reaction; coping; design; dimer; endoribonuclease; expectation; gene function; genetic selection; human DICER1 protein; human disease; improved; inhibitor/antagonist; insight; interest; mRNA Transcript Degradation; molecular recognition; mutant; novel therapeutics; pathogen; plant fungi; public health relevance; reconstitution; research study; tool

DESCRIPTION (provided by applicant): RNA molecules with structure-dependent functions play important roles throughout molecular biology, and the broad, long-term objective of this grant is to understand these roles. The experiments of this proposal focus on the mechanism, biology, and evolution of RNA interference (RNAi), an example of a structure-dependent function in which double-stranded RNA (dsRNA) triggers the destruction of corresponding cellular mRNAs. Although RNAi has been lost in Saccharomyces cerevisiae, it is present in other budding yeasts, including Saccharomyces castellii (a close relative of S. cerevisiae) and Candida albicans (a common human pathogen). These species use noncanonical Dicer proteins to process long dsRNA into small interfering RNAs (siRNAs), which are loaded into the Argonaute protein to direct silencing. Introducing Dicer and Argonaute of S. castellii restores RNAi in S. cerevisiae, and the reconstituted pathway silences endogenous retrotransposons. The discovery of RNAi in budding yeast opens new opportunities for exploring the mechanism, biology, and evolution of the pathway. The first specific aim of the proposed experiments is to determine the consequences of losing or restoring RNAi. Methods will include phenotypic profiling, small-RNA sequencing, and mRNA sequencing. Endogenous dsRNA elements known as Killer elements also will be monitored, with the expectation that their retention will be compromised in the RNAi-reconstituted S. cerevisiae strain. These experiments will explore how cells cope with the introduction of a new gene-regulatory pathway and provide insight into why RNAi was lost in some species. The second aim is to identify additional components of RNAi in budding yeast. Methods will include genetic selections and mass spectrometry of co-immunoprecipitated proteins. Identifying new components and modifiers would be important not only for understanding the yeast pathway but could shed light on RNAi pathways in plants and animals, including humans. Results of both this aim and aim 1 will also be of practical interest for those using RNAi-reconstituted strains to study gene function in S. cerevisiae and those attempting to port RNAi into other RNAi-deficient organisms. The third aim is to determine the mechanism of RNAi in budding yeast. To test the hypothesis that silencing is post- transcriptional, RNAi-mutant strains will be monitored for changes in RNA Polymerase II binding, mRNA turnover, and the correspondence between sequenced siRNAs and mRNA degradation fragments. Biochemical and structural experiments will also test a proposed mechanism for how purified budding-yeast Dicer produces 23-nt siRNAs, despite lacking the protein domain that canonical Dicers require for this activity. Experiments of all three aims will leverage, for the first time, the powerful tools of budding yeast for the study of RNAi. A thorough understanding of this recently identified pathway in budding yeast should provide important insights regarding RNAi and related gene-silencing pathways in other eukaryotes and thereby contribute to fundamental knowledge relevant to human health.

描述（由申请人提供）：具有结构依赖功能的RNA分子在分子生物学中起着重要作用，本资助的广泛、长期目标是了解这些作用。本提案的实验重点是RNA干扰（RNAi）的机制，生物学和进化，这是双链RNA （dsRNA）触发相应细胞mrna破坏的结构依赖功能的一个例子。虽然RNAi在酿酒酵母中缺失，但它存在于其他出芽酵母中，包括酿酒酵母（酿酒酵母的近亲）和白色念珠菌（一种常见的人类病原体）。这些物种使用非规范的Dicer蛋白将长dsRNA加工成小干扰rna (sirna)， sirna被装载到Argonaute蛋白中直接沉默。引入S. castellii的Dicer和Argonaute恢复了S. cerevisiae中的RNAi，重组的途径使内源性反转录转座子沉默。在出芽酵母中发现RNAi为探索该途径的机制、生物学和进化提供了新的机会。提出的实验的第一个具体目的是确定丢失或恢复RNAi的后果。方法包括表型分析、小rna测序和mRNA测序。内源性dsRNA元件也被称为杀伤元件，也将被监测，期望它们的保留将在rnai重组的酿酒葡萄球菌菌株中受到损害。这些实验将探索细胞如何应对新的基因调控途径的引入，并提供RNAi在某些物种中丢失的原因。第二个目标是鉴定出芽酵母中RNAi的附加成分。方法将包括基因选择和共免疫沉淀蛋白的质谱分析。确定新的成分和修饰剂不仅对理解酵母途径很重要，而且可以阐明植物和动物（包括人类）的RNAi途径。这一目的和目的1的结果也将对那些使用RNAi重组菌株来研究酿酒葡萄球菌的基因功能和那些试图将RNAi移植到其他RNAi缺乏的生物体内的人有实际的兴趣。第三个目的是确定RNAi在芽殖酵母中的作用机制。为了验证沉默是转录后的假设，rnai突变株将被监测RNA聚合酶II结合、mRNA周转以及测序的sirna和mRNA降解片段之间的对应关系的变化。生化和结构实验也将测试纯化的发芽酵母Dicer如何产生23-nt sirna的机制，尽管缺乏典型Dicer所需的蛋白质结构域。这三个目标的实验将首次利用出芽酵母的强大工具来研究RNAi。深入了解最近在出芽酵母中发现的这一途径，将为其他真核生物中的RNAi和相关基因沉默途径提供重要见解，从而有助于了解与人类健康相关的基础知识。