MOLECULAR MECHANISM OF DYSKERATOSIS CONGENITA

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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.