Mechanisms of Posttranscriptional Gene Silencing

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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.