Biochemistry Of Small Nucleolar RNAs In Yeast

这项研究的重点是被称为snoRNP的小核仁RNA蛋白机器。大多数snoRNP在rRNA中产生修饰的核苷酸（功能未知）。其他几种促进前体rRNA的切割，可能是通过介导前体rRNA的折叠。存在两种类型的修饰snoRNP，其在特定位点形成2 '-O-甲基化核苷酸（Nm）和假尿苷（psi）。特异性由snoRNA指导序列提供。我们已经确定，rRNA的功能可以通过阻断psi的形成，并显着地，通过引入新的网站Nm修改受损。研究的具体目的是：1）确定snoRNA指导的核苷酸修饰在核糖体RNA中的作用; 2）确定催化活性snoRNA相关蛋白的分子功能; 3）开发snoRNA指导的修饰作为探测RNA结构和功能的策略。核苷酸修饰的作用将通过系统地阻断所选结构域中的修饰，然后评估rRNA成熟、rRNP组装和核糖体功能来确定。待研究的催化蛋白质包括--两种修饰酶，rRNA加工所需的RNA解旋酶，以及snoRNP生物合成所需的两种核质解旋酶。遗传和生化策略将用于确定未知的精确功能，并确定作用机制。为了进一步评估使用snoRNP定向修饰来映射RNA功能的潜力，我们将通过将引导snoRNP文库靶向rRNA中的新结构域来扩展正在进行的rRNA探测研究。几个被认为可以穿过核仁的小RNA也将被靶向，以确定可修饰底物的范围。最后，我们将确定snoRNP定向修饰是否可以阻止致病酵母的生长并破坏HIV复制。

The focus of this research is the small nucleolar RNA protein machines called snoRNPs. Most snoRNPs produce modified nucleotides (of unknown function) in rRNA. A few others facilitate cleavage of pre- rRNA, probably by mediating folding of pre-rRNA. Two types of modifying snoRNPs exist, which form 2'-O-methylated nucleotides (Nm) and pseudouridines (psi) at specific sites. Specificity is provided by snoRNA guide sequences. We have determined that rRNA function can be impaired by blocking psi formation, and remarkably, by introducing Nm modifications at new sites. The specific aims of the research are: 1) to determine the role of snoRNA-directed nucleotide modification in ribosomal RNA; 2) to define the molecular functions of catalytically active snoRNA-associated proteins, and; 3) to develop snoRNA-directed modification as a strategy for probing RNA structure and function in vivo. The role of nucleotide modification will be determined by systematically blocking modifications in selected domains, and then evaluating rRNA maturation, rRNP assembly and ribosome function. The catalytic proteins to be studied include-- the two modifying enzymes, an RNA helicase required for rRNA processing, and two nucleoplasmic helicases required for snoRNP biogenesis. Genetic and biochemical strategies will be used to identify precise functions where unknown, and to define the mechanisms of action. To further assess the potential for using snoRNP- directed modification for mapping RNA function, we will extend an rRNA probing study in progress by targeting libraries of guide snoRNPs to new domains in rRNA. Several small RNAs thought to pass through the nucleolus will also be targeted, to determine the range of modifiable substrates. Finally, we will determine if snoRNP-directed modification can block growth of pathogenic yeast and disrupt HIV replication.