Biochemistry of Small Nucleolar RNAs in Yeast

核仁含有大量小核仁RNA-蛋白质复合物（snoRNP），其修饰rRNA中的核苷酸（大多数snoRNP这样做）或参与前rRNA的加工（切割）。修饰snoRNP形成2 '-O-甲基化核苷酸和假尿苷，主要在核糖体的功能重要区域。这些变化的影响实际上是未知的。处理snoRNP的主要未回答的问题是它们的实际作用。该提案有两个主要目标--旨在回答这些重要问题。两者都建立在以前赠款的基础上。首先，我们发现核糖体反应中心区域的核苷酸修饰的系统性缺失导致翻译缺陷。其次，我们亲和分离了18 S rRNA加工和甲基化所需的保守snoRNP（U14）， 并鉴定了几种新的蛋白质，包括已经与核糖体合成有关的因子。 我们的应用程序的主要目的是：1）确定核苷酸修饰的影响 在mRNA解码中心和其他两个结构域，动态相互作用的tRNA在 确定U14 snoRNP蛋白在rRNA加工中的作用 和修改。核苷酸修饰的影响将通过消耗选择的 从目标区域的修饰和检查核糖体合成和功能。研究 U14 snoRNP最初将集中精力定义新snoRNP组件的作用， 核糖体合成，通过遗传耗竭，突变和亲和分离策略。其他目标 包括描述snoRNP复合物的组装、结构和活性。 这些研究的结果将为核糖体的合成和功能提供有价值的新见解， 并可能揭示snoRNP，衰老和癌症之间的既定联系。

DESCRIPTION (provided by applicant) The nucleolus contains scores of small nucleolar RNA-protein complexes (snoRNPs), which modify nucleotides in rRNA (most snoRNPs do this) or participate in processing (cleavage) of prerRNA. The modifying snoRNPs form 2'-O-methylated nucleotides and pseudouridine, mostly in functionally important regions of the ribosome. The effects of these alterations are virtually unknown. The main unanswered question with the processing snoRNPs is their actual role. The proposal has two main aims -designed to answer these important questions. Both build on results from the previous grant. First, we showed that systematic depletion of nucleotide modifications from the reaction center region of the ribosome causes defects in translation. Second, we have affinity-isolated a conserved snoRNP (U14) required for processing and methylation of 18S rRNA, and identified several novel proteins, including factors already tied to ribosome synthesis. The major aims of our application are: 1) To determine the influence of nucleotide modifications in the mRNA decoding center and two other domains that interact dynamically with tRNA in the decoding region, and; 2) To determine the roles of the U14 snoRNP proteins in rRNA processing and modification. Effects of nucleotide modification will be investigated by depleting selected modifications from the target regions and examining ribosome synthesis and function. Studies of the U14 snoRNP will focus initially on defining the roles of the new snoRNP components in ribosome synthesis, by genetic depletion, mutation and affinity isolation strategies. Other goals include describing the assembly, structure and activity of the snoRNP complex. Results from the studies will yield valuable new insights into ribosome synthesis and function, and could shed light on the basis of established links between snoRNPs, aging and cancer.