Ribosome Biogenesis: Small Nucleolar RNPs

描述(申请人提供)：核糖体生物发生是一种基本的 进程，但对其已知的信息有很大的空白 机制。我们对发现E1/U17的特异性缺失感到兴奋 小核仁RNA(SnoRNA)产生一种独特的Pre-rRNA切割过程 在青蛙卵母细胞中被U17 RNA特异性逆转的表型。U17 RNA是 对18S rRNA的形成是必不可少的。U17 RNA直接相互作用 (补骨脂素-交联物)在核糖体RNA前体中具有意想不到的位点 (前rRNA)在体内。 长期目标：有助于我们理解Small的作用 核糖体形成中的核仁核糖核蛋白颗粒(SnoRNP)，聚焦 首先是U17 SnoRNP。为简洁起见，本文中的“U 17 RNP角色A”指的是 在U17缺失的细胞中，18SRRNA形成受阻的步骤(S)，这 注射野生型U17RNA可逆转其阻断作用。“RP元素”是指 这里是一个U17 RNA片段，它的替换突变干扰了U17 RNP 角色A。“ SpeqfIc的目标：#1)这是U1 7依赖的Pre-rRNA加工步骤(S) 18S rRNA的形成：末端定位和20S的脉冲追逐标记 U17耗尽的青蛙卵母细胞中的前rRNA。#2)哪些是U1 7的反相元素 RNA？：体内U17RNA突变分析。#3)鉴定蛋白质 与U17 RNA特异性地相互作用。哪个U17 RNA RP元件(在AIM中确定 2)与哪种蛋白质特异地相互作用？： 用反义亲和层析法纯化U1 7snoRNP， 生物素化的2-O-甲基寡核苷酸.用MALDI进行蛋白质鉴定 和ESI质谱学，或部分微测序；U17RNA足迹； RNA竞争凝胶迁移率分析与RNA-蛋白质UV 体内和/或体外的交联剂。#4)哪种U17 RNA-蛋白质特异性 相互作用(在目标3中识别LED)涉及U17 RNP角色A？：Point U17 RNARP元件中及其附近的突变：它们是否紧密耦合 对U17RNP角色A和U17RNA-蛋白质相互作用的影响？我们将测试 U17 RNP角色A的模型，集中在这些U17 RNA RP元件和它们的 互动。体内的大部分实验将通过注射到 青蛙卵母细胞。

DESCRIPTION (provided by applicant): Ribosome biogenesis is a fundamental process in the cell but there are major gaps in what is known about its mechanism. We are excited about the finding that specific depletion ofE1/U17 small nucleolar RNA (snoRNA) produces a unique pre-rRNA cleavage processing phenotype that is specifically reversed by U17 RNA in frog oocytes. U17 RNA is essential for 18S rRNA formation. U17 RNA interacts directly (psoralen-crosslinks) with unexpected sites in ribosomal RNA precursor (pre-rRNA) in vivo. Long-term goal: To contribute to our understanding of the roles of small nucleolar ribonucleoprotein particles (snoRNPs) in ribosome formation, focusing first on the U 17 snoRNP. For brevity, "U 17 RNP role A" refers in this text to the step(s) in 18S rRNA formation that is blocked in U17-depleted cells, this block being reversed by injection of wild-type U17 RNA. "RP element" refers here to a U17 RNA segment whose substitution mutation interferes with "U17 RNP role A." SpeqfIc Aims: #1) Which is the U1 7-dependent pre-rRNA processing step(s) in 18S rRNA formation?: Mapping of termini and pulse-chase labeling of "20S" pre-rRNA in U17-depleted frog oocytes. #2) Which are ihe RP elements of U1 7 RNA?: U17 RNA mutation analysis in vivo. #3) Identification ofproteins that interact specifically with U17 RNA. Which U17 RNA RP element (identified in Aim 2) interacts specifically with which protein?: Affinity chromatography purification of the U1 7 snoRNP using antisense, biotinylated, 2 -O-methy1 oligoribonucleotides; protein identification by MALDI and ESI mass spectrometry, or partial microsequencing; U17 RNA footprinting; RNA competition electrophoretic mobility shift assays and RNA-protein UV crosslinking, both in vivo and/or in vitro. #4) Which U17 RNA -protein specific interactions (identify led in Aim 3) are involved in U17 RNP role A?: Point mutations in and next to a U17 RNA RP element: do they have tightly coupled effects on U17 RNP role A and a U17 RNA-protein interaction? We will test models ofUl 7 RNP role A, focused on these U17 RNA RP elements and their interactions. Most of the experiments in vivo will be done by injection into frog oocytes.