THE CONTROL OF GENE EXPRESSION BY EUKARYOTIC RNASE III

细胞RNA的调节周转和加工在细胞生长和分化过程中控制基因表达至关重要。 核糖核酸酶III形成高度特异性的双链RNA（dsRNA）内切核酸酶家族，其控制多种细胞RNA加工途径。 核糖体RNA（rRNA）前体的加工是核糖体RNA（rRNA）前体加工中的重要功能。真核RNA酶III还在大量小核RNA（snRNA）和小核仁RNA（snoRNA）的前体的加工中发挥关键作用，所述小核RNA和小核仁RNA分别对mRNA剪接以及rRNA加工和代谢至关重要。 我们的长期目标是探索RNases III在真核基因表达调控中的多种功能。 色葡萄酿酒酵母RNase III同系物Rnt 1 p将作为模型酶进行研究，以研究双链RNA切割在真核基因表达控制中的功能，并破译dsRNA酶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。彻底鉴定酵母RNase III的天然RNA靶标。 遗传结果表明，真核生物RNases III除了在rRNA、snRNA和snoRNA加工中发挥作用外，还特异性控制几种特定mRNA的水平。 将开发基因组方法来彻底鉴定Rnt 1 p的mRNA底物。 RNA底物的详尽列表将提供关于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识别特定RNA的机制。 寻找Rnt 1 p识别RNA的生物化学决定因素是理解dsRNA内切酶靶向RNA的特异性的重要问题。 使用生物化学，结构和遗传研究的组合，RNA和蛋白质的化学基团，负责RNA识别的真核生物RNase III的特异性将被确定。 3.阐明酵母RNase III活性在细胞代谢中的整合。已经鉴定了几种与Rnt 1 p相互作用的酵母蛋白，并且预期其会影响Rnt 1 p在体内的活性。 将测试这些蛋白质对Rnt 1 p活性和亚细胞定位的影响。 特别是，挑衅性的假设表明，Rnt 1 p的活性是由N-末端乙酰化和Rnt 1 p参与性过程进行了测试。

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.