THE CONTROL OF GENE EXPRESSION BY EUKARYOTIC RNASE III

细胞RNA的调节周转和加工在控制细胞生长和分化过程中的基因表达方面起着关键作用。核糖核酸酶III是一个高度特异的双链RNA(DsRNA)内切酶家族，控制着多种细胞RNA加工途径。原核和真核RNase III在核糖体RNA(RRNA)前体的加工过程中具有保守的重要功能。真核RNase III还在大量小核RNAs和小核仁RNAs前体的加工过程中发挥关键作用，而小核仁RNAs和小核仁RNAs分别是mRNA剪接和rRNA加工和代谢所必需的。我们的长期目标是探索RNase III在控制真核基因表达方面的多重功能。本研究将以酿酒酵母RNase III同源物Rnt1p为模型酶，研究双链RNA裂解在调控真核基因表达中的作用，并破译dsRNAses特异性裂解的生化机制。在这个框架内，我们的具体目标是：1.详尽地鉴定酵母RNase III的天然RNA靶标。遗传结果表明，真核RNase III除了在rRNA、SnRNA和snoRNA加工中发挥作用外，还特异性地控制几种特定mRNAs的水平。将开发基因组学方法来详尽地鉴定Rnt1p的mRNA底物。一份详尽的RNA底物清单将提供有关RNA底物保守结构元件的系统发育信息，并将阐明该酶控制的基因表达途径。2.了解酵母RNase识别RNA的机制III.寻找Rnt1p识别RNA的生化决定因素是了解dsRNA内切酶靶向RNA的特异性的重要问题。利用生化、结构和遗传学的组合研究，将鉴定负责真核RNaseIII识别RNA的特异性的RNA和蛋白质化学基团。3.揭示酵母核糖核酸酶III活性在细胞代谢中的整合。已鉴定出几种与Rnt1p相互作用的酵母蛋白，并有望在体内影响Rnt1p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，关于Rnt1p的活性受N-末端乙酰化调控以及Rnt1p参与性行为过程的挑衅性假设将得到检验。

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.