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NAP 57（又名dyskerin）的基因DKC 1突变引起的。DC是一种经常致命的骨髓衰竭综合征，是一种复杂的多系统疾病。NAP 57与其他三种核心蛋白一起与大约100种不同的小核RNA（snRNA）结合，其特征在于H和ACA基序，以形成尽可能多的核糖核蛋白颗粒（RNP）。这些H/ACA RNP在至少四个不同的核事件中起作用，核糖体RNA（rRNA）假尿苷化、前rRNA加工、剪接体snRNA假尿苷化和端粒酶RNA稳定化。NAP 57作为假尿苷酶起作用，并且是H/ACA RNP的完整性所需的。我们建立了一个体外测定snoRNP介导的假尿苷化，并解决了核心H/ACA RNP的装配图。我们假设NAP 57中的突变不仅影响NAP 57本身，而且影响整个H/ACA RNP及其功能。在这里，我们将测试这一假设，首先，建立一个详细的结构-功能图谱的NAP 57和H/ACA RNP，其次，通过确定DC突变的影响。本研究的主要目的是：（1）通过亲和纯化的方法，从细胞提取物中组装的H/ACA RNP中标记H/ACA RNA，组装并比较四种功能类型的H/ACA RNP;（2）从纯化的重组组分中重组H/ACA RNP，并分析它们与野生型和突变型NAP 57的相互作用;（3）基于细菌同源物的已知结构的NAP 57的三维结构的建模和DC突变的影响的预测;和（4）患者细胞中H/ACA RNP的结构和功能的评估。该应用的结果将允许精确定位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.