Mechanisms of Posttranscriptional Gene Silencing

转录后基因沉默的机制

• 批准号: 6636614
• 负责人: HERIBERTO CERUTTI
• 金额: $ 23.69万
• 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 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6636614
• 关键词: Chlamydomonas; DNA damage; RNA; affinity chromatography; developmental genetics; enzyme activity; gene induction /repression; genetic regulation; genetic transcription; genetically modified plants; helicase; immunocytochemistry; intermolecular interaction; nuclear runoff assay; nucleic acid metabolism; plant genetics; plant proteins; polymerase chain reaction; protein localization; protein purification; protein structure function; site directed mutagenesis; transposon /insertion element; yeast two hybrid system

Epigenetic processes, which result in heritage changes in gene expression without modifications in DNA sequence, play important roles in the control of the development as well as in the cellular responses to viruses, and transposable elements. In fungi, plants, and animals the introduction of transgenes can lead to the silencing of homologous sequences by a variety of epigenetic phenomena operating at both transcriptional and post- transcriptional levels. The long-term goal of our research project is to understand the molecular basis of epigenetic gene silencing in eukaryotic organisms. Our immediate objectives are to identify and characterize genes involved in the post-transcriptional inactivation of transgenes and transposons in the unicellular green alga Chlamydomonas reinhardtii. Tagged and classical mutants defective in post-transcriptional silencing will be isolated by screening for reactivation of expression of a silenced transgene. The corresponding genes will be cloned by a variety of genetic and molecular approaches. The isolated mutants will also be tested for their effects on RNA interference, induced by transgenes designed to produce double-stranded RNA. Proteins interacting with a cloned DEAH- box RNA helicase, that is required for transgene and transposon silencing, will be identified by tandem affinity purification and by using the yeast- two hybrid system. The DEAH-box RNA helicase will be characterized in terms of cytological localization, enzymatic activities, and in vivo role(s) in different gene expression and in the cellular responses to RNA/DNA damage. Our findings will help to elucidate whether this helicase is involved in RNA processing and/or in a putative RNA surveillance system responsible for the degradation of aberrant RNAs. Using mutants defective in the transcriptional or post-transcriptional silencing of transgenes, the effect of these processes on the inactivation of transposable elements will also be evaluated. Transposon transcription will be examined by nuclear run-on assays ans transposon RNA stability will be determined following inhibition of transcription by Actinomycin D> These experiments will provide insights on the possible relationship between the mechanisms responsible for the transcriptional and post-transcriptional silencing. The overall findings are expected to contribute to our understanding of the molecular machinery involved in post-transcriptional gene silencing and RNA interference. If the silencing mechanisms are indeed effective as anti-transposon and anti-viral agents, their further elucidation will have impact not only in basic biology but also in medicine and agriculture.

表观遗传过程导致基因表达的遗传变化而不改变DNA序列，在控制发育以及细胞对病毒和转座因子的反应中发挥重要作用。 在真菌、植物和动物中，转基因的引入可通过在转录和转录后水平上操作的多种表观遗传现象导致同源序列的沉默。 我们的研究项目的长期目标是了解真核生物中表观遗传基因沉默的分子基础。 我们的直接目标是确定和表征基因参与转录后失活的转基因和转座子在单细胞绿色衣藻莱茵衣藻。通过筛选沉默转基因表达的再激活来分离转录后沉默缺陷的标记突变体和经典突变体。 相应的基因将通过各种遗传和分子方法克隆。 还将测试分离的突变体对RNA干扰的影响，RNA干扰由设计用于产生双链RNA的转基因诱导。 与克隆的DEAH盒RNA解旋酶相互作用的蛋白质是转基因和转座子沉默所必需的，将通过串联亲和纯化和使用酵母双杂交系统来鉴定. DEAH盒RNA解旋酶将在细胞学定位、酶活性和不同基因表达中的体内作用以及对RNA/DNA损伤的细胞应答方面进行表征。 我们的研究结果将有助于阐明这种解旋酶是否参与RNA加工和/或负责降解异常RNA的假定RNA监视系统。 使用转基因转录或转录后沉默缺陷突变体，也将评估这些过程对转座因子失活的影响。 转座子转录将通过核连续测定来检查，并且转座子RNA稳定性将在放线菌素D抑制转录后确定。这些实验将提供关于负责转录和转录后沉默的机制之间的可能关系的见解。 整体的研究结果，预计将有助于我们了解的分子机制参与转录后基因沉默和RNA干扰。 如果沉默机制作为抗转座子和抗病毒剂确实有效，那么它们的进一步阐明将不仅在基础生物学上而且在医学和农业上产生影响。