RNA-Mediated Silencing: Mechanisms and Biological Roles in Chlamydomonas

描述（由申请人提供）：RNA介导的过程导致真核生物中基因表达的抑制，并可产生多种结果，如mRNA降解、异染色质形成或DNA甲基化。RNA干扰机制也与microRNA的加工和功能有关，microRNA是一类通过翻译抑制或mRNA切割调节基因表达的小RNA。这些现象在真核生物中的广泛发生表明，它们需要祖先的保守机制，假设在限制寄生元件（如转座子和病毒）的表达以及控制发育程序中发挥重要作用。我们的长期目标是阐明RNA介导的沉默的分子基础。通过使用单细胞衣藻莱茵衣藻作为模型系统，我们已经分离出两类参与RNA沉默的基因的突变体。一组编码似乎直接参与RNAi的因子：Mut 68 p，一种推定的DNA聚合酶β样核苷酸转移酶; MutTOp，一种vasa内含子基因产物的同源物; Mut 91 p，一种新的但进化保守的蛋白质，具有C2 H2锌指和RNA结合基序。另一组基因，典型的Mut 6（编码一个推定的DEAH盒RNA解旋酶），似乎调节前mRNA加工，因此，某些RNAi组件的mRNA水平。这些基因可以调节响应非生物胁迫的RNAi活性。这项建议将侧重于三个主要目标。(1)dsRNA介导的沉默缺陷突变体（Mut-68、Mut-70和Mut-91）的分子表征。所有这些菌株似乎在从长dsRNA到小RNA的加工的下游过程中有缺陷。我们的假设是，这些因素发挥的作用，RISC（RNA引导的核酸内切复合物）的组件，或可能调节其活动/组装和协调切割的成绩单的降解。我们将尝试通过分离与克隆基因产物相互作用的蛋白质，通过测试纯化复合物和重组多肽的生物化学活性，通过检查融合蛋白的亚细胞定位，以及通过与野生型和突变形式的蛋白质互补突变株，然后进行详细的表型和分子表征来定义它们的分子作用。(2)检查RNA沉默在响应非生物胁迫中的生物学作用。这一目标将通过测试突变菌株在不同环境条件下的存活率来实现。将在比较野生型和突变株的微阵列实验中鉴定RNA沉默的潜在靶基因。(3)通过插入诱变筛选分离参与dsRNA介导的基因沉默的其他基因。总体研究结果预计将提高我们利用RNAi作为实验和/或治疗工具的能力，并可能对医学和农业产生影响。

DESCRIPTION (provided by applicant): RNA-mediated processes result in suppression of gene expression in eukaryotes and can produce a variety of outcomes such as mRNA degradation, heterochromatin formation, or DNA methylation. The RNA interference machinery has also been implicated in the processing and function of microRNAs, a class of small RNAs that regulate gene expression by translational repression or mRNA cleavage. The widespread occurrence of these phenomena in eukaryotes suggests that they entail ancestral, conserved mechanisms postulated to play essential roles in limiting the expression of parasitic elements, such as transposons and viruses, as well as in controlling developmental programs. Our long term goal is to elucidate the molecular basis of RNA-mediated silencing. By using the unicellular alga Chlamydomonas reinhardtii as a model system, we have isolated mutants in two classes of genes involved in RNA silencing. One group encodes factors that appear to be directly involved in RNAi: Mut68p, a putative DNA polymerase beta-like nucleotidyltransferase; MutTOp, a homolog of the vasa intronic gene product; and Mut91p, a novel but evolutionary conserved protein with a C2H2 zinc finger and a RNA binding motif. Another group of genes, typified by Mut6 (encoding a putative DEAH-box RNA helicase), seems to regulate the pre-mRNA processing and, thus, the mRNA levels of certain RNAi components. These genes may modulate RNAi activity in response to abiotic stresses. This proposal will focus on three main goals. (1) Molecular characterization of mutants defective in dsRNA-mediated silencing (Mut-68, Mut-70, and Mut-91). All these strains appear to be defective in processes downstream from the processing of long dsRNA to small RNAs. Our hypothesis is that these factors either play a role as components of RISC (the RNA-guided endonucleolytic complex) or may modulate its activity/assembly and coordinate the degradation of cleaved transcripts. We will attempt to define their molecular roles by isolation of proteins interacting with the cloned gene products; by testing the biochemical activity of purified complexes and recombinant polypeptides; by examining the subcellular localization of fusion proteins; and by complementation of mutant strains with wild type and mutated forms of the proteins followed by detailed phenotypic and molecular characterization. (2) Examination of the biological role(s) of RNA-silencing in the response to abiotic stresses. This goal will be achieved by testing the survival of mutant strains under different environmental conditions. Potential target genes of RNA-silencing will be identified in microarray experiments comparing wild-type and mutant strains. (3) Isolation of additional genes involved in dsRNA-mediated gene silencing by insertional mutagenesis screens. The overall findings are expected to improve our ability to exploit RNAi as an experimental and/or therapeutic tool, with likely impacts in both medicine and agriculture.