RNA-Mediated Silencing: Mechanisms and Biological Roles in Chlamydomonas

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DMA甲基化。的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。广泛 这些现象在真核生物中的发生表明它们需要祖先的保守机制 被假定在限制寄生元件如转座子的表达中起重要作用， 病毒，以及控制发育程序。我们的长期目标是阐明 RNA介导的沉默的基础。以单细胞莱茵衣藻为模型， 系统中，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的因素：Mut 68 p，一种假定的DNA聚合酶β-样 核苷酸转移酶; MutTOp，vasa内含子基因产物的同源物;和Mut 91 p，一种新的但 具有C2 H2锌指和RNA结合基序的进化保守蛋白。另一组基因， 以Mut 6（编码一种推定的DEAH盒RNA解旋酶）为代表，似乎调节前mRNA 加工，因此，某些RNAi组分的mRNA水平。这些基因可以调节RNAi 对非生物胁迫的反应。这项建议将侧重于三个主要目标。(1)分子 在一些实施方案中，对dsRNA介导的沉默缺陷的突变体（Mut-68、Mut-70和Mut-91）进行了表征。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，这些因素或者作为RISC的组成部分发挥作用（RNA引导的 核酸内切复合物）或可以调节其活性/组装并协调切割的核酸的降解。 成绩单我们将尝试通过分离与克隆的蛋白质相互作用的蛋白质来确定它们的分子作用。 基因产物;通过测试纯化的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;以及通过突变株与野生型 类型和突变形式的蛋白质，然后详细的表型和分子表征。（二） 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。潜在目标 RNA沉默的基因将在比较野生型和突变株的微阵列实验中鉴定。 (3)通过插入突变分离参与dsRNA介导的基因沉默的其他基因 卡位总体研究结果有望提高我们利用RNAi作为实验和/或 治疗工具，可能对医学和农业产生影响。

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 DMA 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.