Biogenesis of Eukaryotic Ribosomes

真核核糖体的生物发生

• 批准号: 6744084
• 负责人: SUSAN A GERBI
• 金额: $ 26.69万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6744084
• 关键词: DNA footprinting; RNA; Saccharomyces cerevisiae; Xenopus; Xenopus oocyte; antisense nucleic acid; chemical cleavage; chemical structure function; conformation; fungal genetics; fungal proteins; genetic regulatory element; intermolecular interaction; microinjections; molecular genetics; northern blottings; nucleic acid biosynthesis; nucleic acid sequence; nucleic acid structure; nucleolus; polymerase chain reaction; ribosomes; site directed mutagenesis; uracil nucleoside

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.

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 裂解事件。这笔赠款的主要主题是了解 真核 rRNA 加工中的小核仁 RNA (snoRNA)。大部分的 拟议的实验集中于 U3 snoRNA，它是最丰富的 snoRNA，对于 prerRNA 中多个位点的切割至关重要。我们最近的 U3 的解剖已经确定了该分子中具有重要功能的区域。 这里描述的研究将探索 U3 与 pre-rRNA 的相互作用。这 非洲爪蟾卵母细胞将被用作“体内试管”来推断rRNA加工 机制。酵母将用于基因筛选。 (1) U3 snoRNA 5'区点突变分析 完全的;假尿苷（w） Xenopvs U3 snoRNA 在 nt 8 和 12 处的形成将被确认为内源性 U3 并分析注射的 U3 突变体。 (2) 我们最近的结果表明了一组动态碱基对相互作用 U3 snoRNA 和 prerRNA。这些将通过酵母的补偿性变化来评估 和爪蟾。 (3) 进行酵母抑制筛选以鉴定分子间 U3 Box A' 与 RNA 和蛋白质的接触。 (4) 非洲爪蟾 pre-rRNA 的定点诱变将揭示序列和 在位点 AO、I 和 2 处裂解所需的结构。 (5) snoRNA可能在rRNA的生物发生过程中调节其构象。 化学改性 或 U3 snoRNA 耗尽后或耗尽后及随后的 rRNA 前体 注射突变型 U3 snoRNA 将用于分析 U3 snoRNA 是否有影响 前RNA的构象。 (6) 非洲爪蟾卵母细胞中反义寡核苷酸的消耗将证明： 位点 2 和 3 的切割需要 Ui 3 和 7-2IMRP snoRNA， 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，这是核糖体生物发生所必需的基本过程 细胞生长和活力。