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

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基因表达的抑制，并能产生多种

项目成果

A WD40-repeat containing protein, similar to a fungal co-repressor, is required for transcriptional gene silencing in Chlamydomonas.

衣藻转录基因沉默需要含有 WD40 重复序列的蛋白质，类似于真菌共阻遏物。

• DOI: 10.1046/j.1365-313x.2002.01331.x
• 发表时间: 2002
• 期刊: The Plant journal : for cell and molecular biology
• 作者: Zhang,Chaomei;Wu-Scharf,Dancia;Jeong,Byeong-ryool;Cerutti,Heriberto
• 通讯作者: Cerutti,Heriberto

Targeted gene silencing by RNA interference in Chlamydomonas.

• DOI: 10.1016/s0091-679x(08)93005-3
• 发表时间: 2009
• 期刊: Methods in cell biology
• 作者: Eun-jeong Kim;H. Cerutti

Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage.

莱茵衣藻胞质硫氧还蛋白 h1 在响应烷基化诱导的 DNA 损伤中的功能特化。

• DOI: 10.1128/ec.4.2.262-273.2005
• 发表时间: 2005
• 期刊: Eukaryotic cell
• 作者: Sarkar,Nandita;Lemaire,Stephane;Wu-Scharf,Danxia;Issakidis-Bourguet,Emmanuelle;Cerutti,Heriberto
• 通讯作者: Cerutti,Heriberto