Biogenesis of Eukaryotic Ribosomes

描述(申请人摘要)：成熟核糖体RNA的生产 来自-rRNA前体的(RRNA)物种需要有序的序列 乳沟事件。这笔赠款的主要主题是了解 真核rRNA加工中的小核仁RNA(SnoRNAs)。大多数 拟议的实验重点是U3 snoRNA，它是 SnoRNAs，对于切割prerRNA中的几个位点是必不可少的。我们最近 对U3的解剖已经确定了分子中重要的功能区域。 这里描述的研究将探索U3与前rRNA的相互作用。这个 非洲爪哇卵母细胞将被用作“体内试管”来推断rRNA的加工 机械装置。酵母将被用于基因筛查。 (1)将对U3 snoRNA 5‘区点突变进行分析 已完成；假尿苷(Passouridine) Xenopvs U3 snoRNA中第8和12位核苷酸的形成将被确认为内源性 U3，并对注射的U3突变体进行分析。 (2)我们的最新研究结果表明了一组动态碱基相互作用 U3 snoRNA和prerRNA。这些将通过酵母中的代偿性变化来评估 在非洲爪哇。 (3)将进行酵母抑制物筛选以鉴定分子间 U3盒A‘与RNA和蛋白质的接触。 (4)非洲爪哇Pre-rRNA的定点突变将揭示其序列和 A0、I和2位点切割所需的结构。 (5)在rRNA的生物发生过程中，snoRNAs可能调节rRNA的构象。 化学改性 或在U3 snoRNA耗尽后或在耗尽后和随后 注射突变的U3 snoRNA将被用来分析U3 snoRNA是否影响 前-RNA的构象。 (6)非洲爪哇卵母细胞中反义寡核苷酸的耗尽将证明 UI3和7-2IMRP snoRNAs是在位点2和3进行切割所必需的， 分别进行了分析。 拟议的研究应阐明动态rna-rna的重要性。 核糖体生物发生过程中的相互作用，这是 细胞的生长和活力。

DESCRIPTION(applicant's abstract): The production of mature ribosomal RNA (rRNA) species from the- rRNA precursor requires an ordered sequence of cleavage events. The main subject of this grant is to understand the roles of small nucleolar RNAs (snoRNAs) in eukaryotic rRNA processing. Most of the proposed experiments focus on U3 snoRNA, which is the most abundant of the snoRNAs and is essential for cleavage at several sites in prerRNA. Our recent dissection of U3 has identified functionally important areas in the molecule. The studies described here will explore interactions of U3 with pre-rRNA. The Xenopus oocyte will be used as an "in vivo test tube" to deduce rRNA processing mechanisms. Yeast will be used for a genetic screen. (1) Analysis of point mutations in the 5' region of U3 snoRNA will be completed; pseudouridine (w) formation at nt 8 and 12 in Xenopvs U3 snoRNA will be confirmed for endogenous U3 and analyzed for injected U3 mutants. (2) Our recent results suggest a set of dynamic base-pair interactions between U3 snoRNA and prerRNA. These will be assessed by compensatory changes in yeast and in Xenopus. (3) Yeast suppressor screens will be carried out to identify intermolecular contacts of U3 Box A' with RNA and protein. (4) Site-directed mutagenesis of Xenopus pre-rRNA will reveal the sequences and structures required for cleavage at sites AO, I and 2. (5) snoRNAs may modulate the conformation of rRNA during its biogenesis. Chemical modification or pre-rRNAs after depletion of U3 snoRNA or after depletion and subsequent injection of mutant U3 snoRNAs will be used to analyze if U3 snoRNA influences the conformation of pre-RNA. (6) Antisense oligonucleotide depletion in Xenopus oocytes will demonstrate if Ui 3 and 7-2IMRP snoRNAs are required for cleavage at sites 2 and 3, respectively. The proposed studies should elucidate the importance of dynamic RNA-RNA interactions during ribosome biogenesis, a fundamental process essential for cell growth and viability.