RNA Conformation and Catalysis

描述(由申请人提供)：具有结构依赖功能的RNA分子在整个分子生物学中扮演着重要的角色，而这项资助的广泛、长期的目标是了解这些角色。这一建议的实验重点是RNA干扰(RNAi)的机制、生物学和进化，RNAi是双链RNA(DsRNA)触发相应细胞mRNAs破坏的结构依赖功能的一个例子。虽然RNAi已经在酿酒酵母中丢失，但在其他萌芽酵母中也存在，包括酿酒酵母(与酿酒酵母的近亲)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(SiRNAs)，这些小干扰RNAs被加载到ArgAerte蛋白中直接沉默。在酿酒酵母中，通过引入酿酒酵母中的DICER和ARGANOUTE基因，恢复了酿酒酵母中的RNAi，并使内源性反转录转座子沉默。在发芽酵母中发现RNAi为探索该途径的机制、生物学和进化打开了新的机会。拟议实验的第一个具体目标是确定RNAi丢失或恢复的后果。方法包括表型分析、小RNA测序和信使核糖核酸测序。被称为Killer元素的内源性dsRNA元素也将被监测，预计它们在RNAi重组酿酒酵母菌株中的保留将受到影响。这些实验将探索细胞如何应对新的基因调控途径的引入，并为为什么RNAi在某些物种中丢失提供了洞察。第二个目标是在萌芽酵母中鉴定RNAi的其他成分。方法将包括遗传选择和免疫共沉淀蛋白质的质谱分析。识别新的成分和修饰物不仅对了解酵母途径很重要，而且可以揭示包括人类在内的动植物的RNAi途径。这一目标和目标1的结果也将对那些使用RNAi重组菌株来研究酿酒酵母基因功能的人以及那些试图将RNAi转移到其他RNAi缺陷生物中的人具有实际意义。第三个目的是确定发芽酵母中RNAi的机制。为了验证沉默是转录后的假说，将监测RNAi突变株的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。生化和结构实验还将测试一种拟议的机制，即纯化的萌芽酵母DICER如何产生23-NT的siRNAs，尽管缺乏经典的Dicers进行这一活动所需的蛋白质结构域。所有这三个目标的实验都将首次利用发芽酵母这一强大的工具来研究RNAi。对发芽酵母中这一新近发现的途径的彻底理解将为其他真核生物中的RNAi和相关的基因沉默途径提供重要的见解，从而有助于了解与人类健康相关的基础知识。

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.