Mechanisms of Posttranscriptional Gene Silencing

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元件的反应中发挥着重要作用。在真菌、植物和动物中，转基因的引入可以通过在转录和转录后水平上操作的各种表观遗传现象导致同源序列的沉默。我们研究项目的长期目标是了解真核生物表观遗传基因沉默的分子基础。我们的近期目标是鉴定和鉴定与单细胞绿藻莱茵衣藻中转基因和转座子转录后失活相关的基因。在转录后沉默中有缺陷的标记突变体和经典突变体将通过筛选沉默的转基因表达的重新激活来分离。相应的基因将通过各种遗传和分子方法进行克隆。分离的突变体还将测试它们对RNA干扰的影响，这种干扰是由旨在产生双链RNA的转基因诱导的。与克隆的Deah-box 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酵母双杂交系统进行鉴定。Deah-box RNA解旋酶将在细胞学定位、酶活性以及在不同基因表达和细胞对RNADNA损伤的反应中的作用(S)方面进行表征。我们的发现将有助于阐明该解旋酶是否参与了RNA加工和/或可能的RNA监控系统，该系统负责异常RNA的降解。利用在转录或转录后沉默转基因方面有缺陷的突变体，也将评估这些过程对转座元件失活的影响。转座子转录将通过核连续试验进行检测，转座子RNA的稳定性将在放线菌素D和GT抑制转录后确定；这些实验将为转录沉默和转录后沉默机制之间的可能关系提供见解。这一总体发现有望有助于我们理解转录后基因沉默和RNA干扰所涉及的分子机制。如果沉默机制确实是有效的抗转座子和抗病毒药物，它们的进一步阐明不仅将对基础生物学产生影响，而且将对医学和农业产生影响。

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.