RNA-Mediated Silencing: Mechanisms and Biological Roles in Chlamydomonas

RNA 介导的沉默：衣藻中的机制和生物学作用

• 批准号: 7529203
• 负责人: HERIBERTO CERUTTI
• 金额: $ 25.32万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7529203
• 关键词: Affinity Chromatography; Agriculture; Algae; Alleles; Animals; Applications Grants; Biochemical; Biological; Biological Models; Biology; Boxing; C2H2 Zinc Finger; Chimeric Proteins; Chlamydomonas; Chlamydomonas reinhardtii; Cleaved cell; Complementary RNA; Complex; DNA Polymerase beta; DNA-Directed DNA Polymerase; Development; Developmental Process; Double-Stranded RNA; Drosophila genus; Elements; Eukaryota; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Screening; Genomics; Goals; Green Algae; Guide RNA; Heart; Heterochromatin; Homologous Gene; Insertional Mutagenesis; Knowledge; Mediating; Medicine; Messenger RNA; Methylation; MicroRNAs; Molecular; Mutate; Names; Outcome; Pathway interactions; Plants; Play; Process; Protein Family; Proteins; RNA Binding; RNA Degradation; RNA Helicase; RNA Interference; RNA Recognition Motif; RNA-Induced Silencing Complex; Recombinants; Research Personnel; Role; Small Interfering RNA; Small RNA; Stress; Testing; Therapeutic; Transcript; Translational Repression; Virus; Yeasts; base; endonuclease; fungus; human DICER1 protein; improved; mRNA Precursor; mRNA Transcript Degradation; member; mutant; novel; nucleotidyltransferase; polypeptide; programs; research study; response; tool; yeast two hybrid system; Affinity Chromatography; Agriculture; Algae; Alleles; Animals; Applications Grants; Biochemical; Biological; Biological Models; Biology; Boxing; C2H2 Zinc Finger; Chimeric Proteins; Chlamydomonas; Chlamydomonas reinhardtii; Cleaved cell; Complementary RNA; Complex; DNA Polymerase beta; DNA-Directed DNA Polymerase; Development; Developmental Process; Double-Stranded RNA; Drosophila genus; Elements; Eukaryota; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Screening; Genomics; Goals; Green Algae; Guide RNA; Heart; Heterochromatin; Homologous Gene; Insertional Mutagenesis; Knowledge; Mediating; Medicine; Messenger RNA; Methylation; MicroRNAs; Molecular; Mutate; Names; Outcome; Pathway interactions; Plants; Play; Process; Protein Family; Proteins; RNA Binding; RNA Degradation; RNA Helicase; RNA Interference; RNA Recognition Motif; RNA-Induced Silencing Complex; Recombinants; Research Personnel; Role; Small Interfering RNA; Small RNA; Stress; Testing; Therapeutic; Transcript; Translational Repression; Virus; Yeasts; base; endonuclease; fungus; human DICER1 protein; improved; mRNA Precursor; mRNA Transcript Degradation; member; mutant; novel; nucleotidyltransferase; polypeptide; programs; research study; response; tool; yeast two hybrid system

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.

RNA介导的过程导致真核生物中基因表达的抑制，并且可以产生一种多样性 诸如mRNA降解，异染色质形成或DMA甲基化之类的结果。 RNA 干涉机械也与MicroRNA的处理和功能有关，这是一类 小的RNA通过翻译抑制或mRNA裂解来调节基因表达。广泛的 这些现象在真核生物中的发生表明它们需要祖先保守的机制 假设是在限制寄生元件的表达中发挥重要作用，例如转座和 病毒以及控制发展计划。我们的长期目标是阐明分子 RNA介导的沉默的基础。通过使用单细胞藻类衣原体Reinhardtii作为模型 系统，我们在参与RNA沉默的两类基因中具有孤立的突变体。一个组编码 RNAi直接参与RNAi的因素：MUT68P，一种推定的DNA聚合酶β样的因素 核苷转移酶； Muttop，VASA内含子基因产物的同源物；和mut91p，一部小说，但 带有C2H2锌指和RNA结合基序的进化保守蛋白。另一组基因， 以mut6为代表（编码假定的DEAH-box RNA解旋酶），似乎调节了前MRNA 处理某些RNAi成分的mRNA水平。这些基因可能调节RNAi 响应非生物应力的活性。该提案将重点介绍三个主要目标。 （1）分子 DSRNA介导的沉默（MUT-68，MUT-70和MUT-91）中有缺陷的突变体的表征。所有这些 从长dsRNA到小RNA的处理过程中，菌株似乎在下游过程中有缺陷。 我们的假设是，这些因素要么作为RISC的组成部分起作用（RNA引导 内核酸化复合物）或可能调节其活性/组装并协调裂解的降解 成绩单。我们将尝试通过隔离与克隆的蛋白质来定义它们的分子作用 基因产物；通过测试纯化的复合物和重组多肽的生化活性；经过 检查融合蛋白的亚细胞定位；并通过与野生菌株的补充 蛋白质的类型和突变形式，然后是详细的表型和分子表征。 （2） 检查RNA - 分解在对非生物应激的反应中的生物学作用。这个目标将是 通过在不同环境条件下测试突变菌株的存活而实现。潜在目标 在比较野生型和突变菌株的微阵列实验中，将确定RNA沉默的基因。 （3）通过插入诱变分离参与DSRNA介导的基因沉默的其他基因 屏幕。预计总体发现将提高我们利用RNAi作为实验和/或的能力 治疗工具，可能对医学和农业产生影响。