Ribosome Biogenesis: Small Nucleolar RNPs

描述（由申请人提供）：核糖体生物发生是一种基本的 细胞中的过程，但在已知的关于其 机制我们对E1/U17特异性耗尽的发现感到兴奋 小核仁RNA（snoRNA）产生独特的前rRNA切割过程 在青蛙卵母细胞中，U17 RNA特异性逆转了这一表型。U17 RNA 18 S rRNA的形成。U17 RNA直接相互作用 核糖体RNA前体中具有非预期位点的（蛋白酶-交联） （pre-rRNA）。 长期目标：促进我们对小企业 核仁核糖核蛋白颗粒（snoRNP）在核糖体形成中的作用，聚焦 首先是U17 snoRNP。为简洁起见，“U 17 RNP角色A”在本文中是指 在U17缺失细胞中被阻断的18 S rRNA形成步骤， 阻断被注射野生型U17 RNA逆转。“RP元件”是指 此处是U17 RNA片段，其取代突变干扰“U17 RNP 角色A. " SpeqfIc目的：#1）哪一个是U1 7依赖性前rRNA加工步骤， 18 S rRNA形成？：“20 S”末端定位和脉冲追踪标记 前体rRNA在去除U17的青蛙卵母细胞中的表达。#2）U1 7的RP元素是什么 RNA？：体内U17 RNA突变分析。#3）鉴定蛋白质， 与U17 RNA特异性相互作用。哪种U17 RNA RP元件（在Aim中鉴定） 2)与哪种蛋白质发生特异性相互作用？ 使用反义、亲和层析纯化U1 7 snoRNP 生物素化的2 -O-甲基寡核糖核苷酸; MALDI法鉴定蛋白质 和ESI质谱法，或部分微测序; U17 RNA足迹; RNA竞争电泳迁移率变动分析和RNA-蛋白质UV 交联，在体内和/或体外。#4）哪种U17 RNA -蛋白质特异性 U17 RNP角色A中涉及的相互作用（在目标3中识别领导）：点 U17 RNA RP元件中及其附近的突变：它们是否与 对U17 RNP作用A和U17 RNA-蛋白质相互作用的影响？我们将测试 U17 RNP角色A的模型，集中于这些U17 RNA RP元件及其 交互.大多数体内实验将通过注射到 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.