Biogenesis of Eukaryotic Ribosomes

描述（申请人摘要）：成熟核糖体RNA的产生 来自- rRNA前体的rRNA（rRNA）种类需要以下的有序序列： 卵裂事件该补助金的主要目的是了解 小核仁RNA（snoRNA）在真核生物rRNA加工中的作用。大部分 建议的实验集中在U3 snoRNA上，这是最丰富的 snoRNA和必需的切割在几个位点的前rRNA。我们最近 对U3的解剖已经确定了分子中的重要功能区域。 本文描述的研究将探索U3与pre-rRNA的相互作用。的 非洲爪蟾卵母细胞将被用作“体内试管”来推断rRNA加工 机制等酵母菌将用于基因筛选。 (1)将对U3 snoRNA的5'区域中的点突变进行分析。 已完成;假尿苷（w） 在Xenopvs U3 snoRNA中在nt 8和12处的形成将被证实是内源性的。 U3，并分析注射的U3突变体。 (2)我们最近的研究结果表明，一组动态碱基对之间的相互作用， U3 snoRNA和prerRNA。这些将通过酵母的代偿性变化来评估 和非洲爪蟾。 (3)将进行酵母抑制剂筛选以鉴定分子间 U3盒A'与RNA和蛋白质的接触。 (4)非洲爪蟾前rRNA的定点诱变将揭示序列， 在位点AO、I和2处切割所需的结构。 (5)snoRNA在rRNA的生物合成过程中可以调节rRNA的构象。 化学修饰 或在消耗U3 snoRNA后或在消耗和随后的 注射突变的U3 snoRNA将用于分析U3 snoRNA是否影响 前体RNA的构象。 (6)爪蟾卵母细胞中的反义寡核苷酸耗竭将证明， Ui 3和7- 2 IMRP snoRNA是在位点2和3处切割所需的， 分别 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，这是一个基本的过程， 细胞生长和活力。

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.