Biochemistry of Small Nucleolar RNAs in Yeast

描述(申请人提供)核仁包含数十个小的核仁RNA-蛋白质复合体(SnoRNPs)，它们改变rRNA中的核苷酸(大多数snoRNPs做这项工作)或参与加工(切割)prerRNA。修饰的snoRNPs形成2‘-O-甲基化的核苷酸和假尿苷，主要位于核糖体的重要功能区域。这些变化的影响几乎是未知的。处理snoRNP的主要悬而未决的问题是它们的实际作用。该提案有两个主要目的--旨在回答这些重要问题。两者都是在前一次拨款结果的基础上发展起来的。首先，我们证明了核糖体反应中心区域的核苷酸修饰的系统性耗尽会导致翻译缺陷。其次，我们亲和地分离了18S rRNA加工和甲基化所需的保守的snoRNP(U14)， 并确定了几种新的蛋白质，包括已经与核糖体合成相关的因子。 我们应用的主要目的是：1)确定核苷酸修饰的影响 在mRNA解码中心和另外两个与tRNA动态相互作用的区域中 解码区，以及；2)确定U14 snoRNP蛋白在rRNA加工中的作用 和改装。核苷酸修饰的效果将通过消耗选定的 从靶区进行修饰，并检测核糖体的合成和功能。的研究 U14 snoRNP最初将专注于定义新snoRNP组件在 核糖体合成，通过基因耗尽、突变和亲和力分离策略。其他目标 包括描述snoRNP复合体的组装、结构和活性。 研究结果将对核糖体的合成和功能产生有价值的新见解， 并可能阐明snoRNPs、衰老和癌症之间已建立的联系。

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.