Biochemistry Of Small Nucleolar RNAs In Yeast

这项研究的重点是被称为snoRNPs的小核仁RNA蛋白质机器。大多数snoRNP在rRNA中产生修饰的核苷酸(功能未知)。其他一些促进前-rRNA的切割，可能是通过调节前-rRNA的折叠。存在两种类型的修饰snoRNPs，它们在特定的位点形成2‘-O-甲基化核苷酸(Nm)和假尿苷(Psi)。特异性由snoRNA引导序列提供。我们已经确定，通过阻止psi的形成，以及在新的位点引入Nm修饰，rRNA的功能可以受到损害。本研究的具体目的是：1)确定snoRNA在核糖体RNA中的作用；2)确定具有催化活性的snoRNA相关蛋白的分子功能；3)开发snoRNA定向修饰作为探测体内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选定结构域的修饰，然后评估rRNA成熟、rRNP组装和核糖体功能来确定。要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生物发生所需的两种核质解旋酶。遗传和生化策略将被用来确定未知的精确功能，并定义作用机制。为了进一步评估使用snoRNP指导的修饰来定位RNA功能的可能性，我们将扩展一项正在进行的rRNA探测研究，将引导snoRNP的文库靶向rRNA中的新结构域。被认为穿过核仁的几个小RNA也将成为靶标，以确定可修饰底物的范围。最后，我们将确定snoRNP定向的修饰是否可以阻止致病酵母的生长和干扰HIV的复制。

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.