MOLECULAR MECHANISM OF DYSKERATOSIS CONGENITA

描述(由申请人提供)： X-连锁先天性角化不良(DC)是由编码NAP57(又名dyskerin)的基因DKC1突变引起的。DC是一种通常致命的骨髓衰竭综合征，是一种复杂的多系统疾病。NAP57和其他三种核心蛋白与100多个以H和ACA基序为特征的不同小核RNA(SnRNAs)结合形成相同数量的核糖核蛋白(RNPs)。这些H/ACA RNPs至少在四个不同的核事件中发挥作用，即核糖体RNA(RRNA)假尿化、前rRNA加工、剪接体SnRNA假尿化和端粒酶RNA稳定。NAP57作为假尿苷酸酶发挥作用，是H/ACA RNPs完整性所必需的。我们建立了snoRNP介导的假尿嘧啶核苷的体外检测方法，并解析了核心H/ACA RNPs的组装图。我们假设NAP57的突变不仅影响NAP57本身，而且影响整个H/ACA RNP及其功能(S)。在这里，我们将首先建立NAP57和H/ACA RNPs的详细结构-功能图谱，然后通过确定DC突变的影响来检验这一假说。这将在以下四个具体目标中进行探讨：(1)通过细胞提取物中组装的H/ACA RNPs的标记H/ACA RNA来亲和纯化H/ACA RNPs的四个功能类别；(2)利用纯化的重组成分重组H/ACA RNPs和野生型和突变型NAP57，并分析它们之间的相互作用；(3)基于已知的细菌同源物的结构和预测DC突变的影响，对NAP57的三维结构进行建模；以及(4)在患者细胞中评估H/ACA RNPs的结构和功能。这一应用的结果将使人们能够准确地确定DC突变对几种基本细胞功能的分子后果，从而为小分子/药物的开发提供基础。

DESCRIPTION (provided by applicant): X-linked dyskeratosis congenita (DC) is caused by mutations in DKC1, the gene encoding NAP57 (aka dyskerin). DC is an often-fatal bone marrow failure syndrome and a complex multi-system disorder. NAP57 together with three other core proteins associates with one hundred or so different small nuclear RNAs (snRNAs) characterized by H and ACA motifs to form as many ribonucleoprotein particles (RNPs). These H/ACA RNPs function in at least four distinct nuclear events, ribosomal RNA (rRNA) pseudouridylation, pre-rRNA processing, spliceosomal snRNA pseudouridylation, and telomerase RNA stabilization. NAP57 functions as the pseudouridylase and is required for the integrity of H/ACA RNPs. We established an in vitro assay for snoRNP-mediated pseudouridylation and resolved an assembly map for core H/ACA RNPs. We hypothesize that mutations in NAP57 not only affect NAP57 itself but also the entire H/ACA RNP and its function(s). Here we will test this hypothesis by, first, establishing a detailed structure-function map of NAP57 and H/ACA RNPs and, second, by determining the impact of DC mutations. This will be approached in the following four Specific Aims: (1) assembly and comparison of the four functional classes of H/ACA RNPs by affinity purification via tagged H/ACA RNAs of H/ACA RNPs assembled in cell extracts; (2) reconstitution of H/ACA RNPs with wild type and mutant NAP57 from purified recombinant components and analysis of their interactions; (3) modeling of the three-dimensional structure of NAP57 based on the known structure of bacterial homologs and prediction of the impact of DC mutations; and (4) evaluation of structure and function of H/ACA RNPs in patient cells. The results of this application will allow pinpointing the molecular consequences of DC mutations on several basic cellular functions and thereby provide the basis for small molecule/drug development.