RNA-Mediated Silencing: Mechanisms and Biological Roles in Chlamydomonas

描述(申请人提供)：RNA介导的过程导致真核细胞中基因表达的抑制，并可产生多种结果，如mRNA降解、异染色质形成或DNA甲基化。RNA干扰机制也参与了microRNAs的处理和功能，microRNAs是一类通过翻译抑制或mRNA切割来调节基因表达的小RNA。这些现象在真核生物中的广泛存在表明，它们与祖先保守的机制有关，这些机制被认为在限制转座子和病毒等寄生成分的表达以及控制发育程序方面发挥着至关重要的作用。我们的长期目标是阐明RNA介导沉默的分子基础。以单细胞藻类莱茵衣藻为模型系统，我们从两类参与RNA沉默的基因中分离出突变体。一组编码似乎直接参与RNAi的因子：Mut68p，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；以及Mut91p，一种具有C2H2锌指和RNA结合基序的新型进化保守蛋白。另一组基因，以Mut6(编码一个假定的Deah-box RNA解旋酶)为代表，似乎调节着mRNA的前加工，从而调节某些RNAi成分的mRNA水平。这些基因可能调节RNAi活性以响应非生物胁迫。这项提案将集中于三个主要目标。(1)dsRNA介导沉默缺陷突变体(Mut-68、Mut-70和Mut-91)的分子特征。所有这些菌株在从长dsRNA到小RNA的下游加工过程中似乎都存在缺陷。我们的假设是，这些因子要么作为RISC(RNA引导的核酸内切复合体)的组成部分发挥作用，要么可能调节其活性/组装并协调切割的转录本的降解。我们将试图通过分离与克隆的基因产物相互作用的蛋白质，通过测试纯化的复合体和重组多肽的生化活性，通过检查融合蛋白的亚细胞定位，通过突变菌株与野生型和突变形式的蛋白质互补，然后详细的表型和分子特征，来确定它们的分子作用。(2)研究RNA沉默在非生物胁迫反应中的生物学作用(S)。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。在比较野生型和突变型菌株的微阵列实验中，将识别潜在的RNA沉默目标基因。(3)通过插入诱变筛选分离与dsRNA介导的基因沉默相关的额外基因。总体研究结果有望提高我们利用RNAi作为实验和/或治疗工具的能力，这可能会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.