Biogenesis of H/ACA Ribonucleoproteins

H/ACA 核糖核蛋白的生物发生

• 批准号: 9189073
• 负责人: U THOMAS MEIER
• 金额: $ 7.93万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9189073
• 关键词: Biogenesis; ACA; Ribonucleoproteins

DESCRIPTION (provided by applicant): Human H/ACA ribonucleoproteins (RNPs) are important for many basic cellular processes including protein synthesis, pre-mRNA splicing, and genome integrity. Among a growing number of functions, H/ACA RNPs isomerize some 130 uridines to pseudouridines in ribosomal (r) and spliceosomal small nuclear (sn) RNAs, process rRNA, stabilize telomerase RNA, and yield microRNAs. Each of these functions is specified by one of over 150 unique H/ACA RNAs, each of which associates with the same four core proteins to form an H/ACA RNP. The central core protein NAP57 (aka dyskerin) is mutated in the predominant X-linked form of the inherited bone marrow failure syndrome dyskeratosis congenita. Although consisting of only five components, biogenesis of these particles is surprisingly complex requiring at least four assembly factors, SHQ1, NAF1, and pontin and reptin. Our recent demonstration that dyskeratosis congenita mutations in NAP57 modulate its interaction with SHQ1 marks DC as an RNP assembly deficiency. This proposal will elucidate mechanisms of assembly factor function in H/ACA RNP biogenesis and thereby not only advance the field but also inform on the basic mechanism of a human disease. This will be approached in the following four Aims: by defining (i) the function of SHQ1 vis-`-vis NAP57, (ii) the role of pontin and reptin in SHQ1 removal from NAP57, (iii) the function of NAF1 in H/ACA RNP biogenesis, and (iv) by identifying the full complement for H/ACA RNP assembly factors. To achieve these goals, we will develop novel approaches, such as dual-color fluorescence fluctuation spectroscopy for the study of protein-protein interaction in living cells, and rely on our established assay systems, such as in vitro assembly of functional H/ACA RNPs in cytosolic extracts and in vivo assembly in our H/ACA RNA inducible cell line. These studies are intended to work towards our long-term goals to define all H/ACA RNP assembly factors for in vitro reconstitution of functional RNPs from recombinant components, to characterize the molecular basis of dyskeratosis congenita by comparing wild type and mutant particles, to obtain high resolution structures of H/ACA RNPs, and to spatially define the process of H/ACA RNP biogenesis relative to subcellular location.

描述（由申请人提供）：人类H/ACA核糖核蛋白（RNPs）对许多基本细胞过程很重要，包括蛋白质合成，mrna前剪接和基因组完整性。在越来越多的功能中，H/ACA RNPs在核糖体(r)和剪接体小核（sn） RNA中将大约130种尿嘧啶异构为伪尿嘧啶，加工rRNA，稳定端粒酶RNA，并产生微小RNA。每一种功能都是由150多个独特的H/ACA rna中的一个指定的，每一个都与相同的四个核心蛋白结合形成H/ACA RNP。核心蛋白NAP57（又名dyskerin）在遗传性骨髓衰竭综合征先天性角化异常的主要x连锁形式中发生突变。尽管这些颗粒仅由五种组分组成，但其生物发生却异常复杂，至少需要四种组装因子：SHQ1、NAF1、pontin和reptin。我们最近的研究表明，NAP57的先天性角化不良突变调节了它与SHQ1的相互作用，这标志着DC是RNP组装缺陷。本研究将阐明装配因子在H/ACA RNP生物发生中的作用机制，不仅有助于该领域的发展，而且有助于了解人类疾病的基本机制。这将通过以下四个目标来实现：通过定义(i) SHQ1对NAP57的功能，（ii）桥蛋白和reptin在从NAP57中去除SHQ1中的作用，（iii） NAF1在H/ACA RNP生物发生中的功能，以及（iv）通过确定H/ACA RNP组装因子的完整补体。为了实现这些目标，我们将开发新的方法，例如用于研究活细胞中蛋白质-蛋白质相互作用的双色荧光波动光谱，并依赖于我们已经建立的分析系统，例如在胞质提取物中体外组装功能性H/ACA RNPs，以及在我们的H/ACA RNA诱导细胞系中体内组装。这些研究旨在实现我们的长期目标，确定所有H/ACA RNP组装因子，用于从重组成分中体外重建功能性RNP，通过比较野生型和突变型颗粒来表征先天性角化不良的分子基础，获得H/ACA RNP的高分辨率结构，并在空间上确定H/ACA RNP的生物发生过程相对于亚细胞位置。