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 例如，mRNA降解、异染色质形成或DMA甲基化。核糖核酸 干扰机制也与microRNAs的处理和功能有关，microRNAs是一类 通过翻译抑制或信使核糖核酸切割来调节基因表达的小RNA。广为流传 这些现象在真核生物中的出现表明它们与祖先的保守机制有关。 被认为在限制寄生成分的表达方面发挥重要作用，如转座子和 病毒，以及在控制发育程序方面。我们的长期目标是阐明分子 RNA介导沉默的基础。以单细胞藻类莱茵衣藻为模型 系统中，我们已经在两类与RNA沉默有关的基因中分离到突变。一组编码 似乎与RNAi直接相关的因素：Mut68p，一种可能的DNA聚合酶样β 核苷酸转移酶；MutTOp，VasA内含子基因产物的同源物；以及Mut91p，一种新的但 具有C2H2锌指和RNA结合基序的进化保守蛋白质。另一组基因， 以Mut6(编码一个假定的Deah-box RNA解旋酶)为代表，似乎调节Pre-mRNA 加工，因此，某些RNAi组件的mRNA水平。这些基因可能调节RNAi 响应非生物胁迫的活动。这项提案将集中于三个主要目标。(1)分子 DsRNA介导的沉默缺陷突变株(Mut-68、Mut-70和Mut-91)的特征。所有这些都是 菌株似乎在从长dsRNA加工到小RNA加工的下游过程中存在缺陷。 我们的假设是，这些因素要么作为RISC(RNA引导的)的组成部分发挥作用 核酸内切复合体)或可调节其活性/组装并协调裂解的降解 成绩单。我们将试图通过分离与克隆的蛋白质相互作用来确定它们的分子作用 基因产品；通过检测纯化的复合体和重组多肽的生化活性； 检测融合蛋白的亚细胞定位；并通过突变株与野生株的互补 蛋白质的类型和突变形式，然后是详细的表型和分子特征。(2) 核糖核酸沉默在响应非生物胁迫中的生物学作用的研究(S)。这个目标将是 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沉默的基因将通过比较野生型和突变菌株的微阵列实验来识别。 (3)插入诱变法分离dsRNA介导的基因沉默相关基因 屏幕。总体结果有望提高我们利用RNAi作为实验和/或 治疗工具，在医学和农业方面都可能产生影响。

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.