THE CONTROL OF GENE EXPRESSION BY EUKARYOTIC RNASE III

调控细胞rna的周转和加工在细胞生长和分化过程中对基因表达的控制至关重要。核糖核酸酶III形成了一个高度特异性的双链RNA （dsRNA）内切酶家族，它控制着多种细胞RNA加工途径。原核生物和真核生物的rnase III在核糖体RNA （rRNA）前体的加工中具有保守的重要功能。真核生物rnase III还在小核rna （snrna）和小核仁rna （snoRNAs）的大量前体的加工中发挥关键作用，这些前体分别对mRNA剪接和rRNA加工和代谢至关重要。我们的长期目标是探索RNases III在控制真核生物基因表达中的多种功能。本研究将以酿酒酵母RNase III同源物Rnt1p为模型酶，研究双链RNA切割在真核基因表达调控中的作用，并解读dsrnase特异性切割的生化机制。在这个框架内，我们的具体目标是：1。全面鉴定酵母RNase III的天然RNA靶点。遗传学结果表明，真核生物RNases III除了在rRNA、snRNA和snoRNA加工中发挥作用外，还能特异性控制几种特定mrna的水平。将开发基因组方法来详尽地鉴定Rnt1p的mRNA底物。详尽的RNA底物列表将提供关于RNA底物的保守结构元件的系统发育信息，并将阐明由该酶控制的基因表达途径。2. 了解酵母RNase III特异性RNA识别的机制。寻找Rnt1p识别RNA的生化决定因素是了解dsRNA内切酶靶向RNA特异性的重要问题。结合生物化学、结构和遗传学研究，将确定真核生物RNase III对RNA识别特异性负责的RNA和蛋白质化学基团。3. 揭示酵母RNase III活性在细胞代谢中的整合。已经确定了几种酵母蛋白与Rnt1p相互作用，并有望影响Rnt1p在体内的活性。这些蛋白对Rnt1p活性和亚细胞定位的影响将被测试。特别是，Rnt1p活性受N端乙酰化调节以及Rnt1p参与性过程的煽动性假设将得到验证。

Regulated turnover and processing of cellular RNAs is critical in the control of gene expression during cell growth and differenciation. Ribonucleases III form a family of highly- specific double-stranded RNA (dsRNA) endonucleases which control a wide variety of cellular RNA processing pathways. Prokaryotic and eukaryotic RNases III have a conserved important function in the processing of the precursor of ribosomal RNA (rRNA). Eukaryotic RNases III also play critical roles in the processing of a large number of precursors of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs), which are essential to mRNA splicing, and rRNA processing and metabolism, respectively. Our long term goal is to explore the multiple functions of RNases III in the control of eukaryotic gene expression. The S. cerevisiae RNase III homolog Rnt1p will be studied as a model enzyme to investigate the function of double-stranded RNA cleavage in the control of eukaryotic gene expression, and to decipher the biochemical mechanisms of specific cleavage by dsRNAses. Within this framework, our specific aims are: 1. To exhaustively identify natural RNA targets of yeast RNase III. Genetic results indicate that eukaryotic RNases III specifically controls the level of several specific mRNAs, in addition to their roles in rRNA, snRNA and snoRNA processing. Genomic approaches will be developed to exhaustively identify mRNA substrates of Rnt1p. An exhaustive list of RNA substrates will provide the phylogenetic information regarding conserved structural elements of the RNA substrates, and will shed light on the gene expression pathways controlled by this enzyme. 2. To understand the mechanisms of specific RNA recognition by yeast RNase III. Finding the biochemical determinants of RNA recognition by Rnt1p is an important issue to understand the specificity of RNA targeting by dsRNA endonucleases. Using a combination of biochemical, structural and genetic studies, RNA and protein chemical groups which are responsible for the specificity of RNA recognition by eukaryotic RNase III will be identified. 3. To unravel the integration of yeast RNase III activity in the cell metabolism. Several yeast proteins have been identified which interact with Rnt1p and are expected to influence Rnt1p activity in vivo. The influence of these proteins on Rnt1p activity and subcellular localization will be tested. In particular, provocative hypotheses suggesting that Rnt1p activity is modulated by N- terminal acetylation and that Rnt1p is involved in the sexual process will be tested.