Biogenesis of H/ACA Ribonucleoproteins

描述（由申请人提供）：人H/ACA核糖核蛋白（RNP）对许多基本细胞过程（包括蛋白质合成、前mRNA剪接和基因组完整性）很重要。在越来越多的功能中，H/ACA RNP将核糖体（r）和剪接体小核（sn）RNA中的约130个尿苷异构化为假尿苷，加工rRNA，稳定端粒酶RNA，并产生microRNA。这些功能中的每一个都由超过150个独特的H/ACA RNA中的一个指定，每个RNA与相同的四个核心蛋白结合形成H/ACA RNP。中心核心蛋白NAP 57（又名dyskerin）在遗传性骨髓衰竭综合征先天性角化不良的主要X连锁形式中发生突变。虽然只有五个组成部分，这些颗粒的生物发生是令人惊讶的复杂，需要至少四个组装因子，SHQ 1，NAF 1，桥蛋白和reptin。我们最近证明NAP 57中的先天性角化不良突变调节其与SHQ 1的相互作用，这标志着DC是RNP组装缺陷。这一建议将阐明组装因子在H/ACA RNP生物发生中的功能机制，从而不仅推进了该领域，而且还为人类疾病的基本机制提供了信息。这将在以下四个目标中进行：通过定义（i）SHQ 1维斯维斯NAP 57的功能，（ii）桥蛋白和reptin在从NAP 57去除SHQ 1中的作用，（iii）NAF 1在H/ACA RNP生物发生中的功能，和（iv）通过鉴定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，如双色荧光波动光谱法用于活细胞中蛋白质-蛋白质相互作用的研究，并依赖于我们建立的测定系统，如在胞质提取物中体外组装功能H/ACA RNP和在我们的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，以确定所有的H/ACA RNP组装因子的功能RNP从重组组件的体外重建，以表征先天性角化不良的分子基础，通过比较野生型和突变体颗粒，以获得高分辨率的结构的H/ACA RNP，并在空间上定义的过程中的H/ACA RNP生物合成相对于亚细胞位置。

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.