Cellular impact of X-linked dyskeratosis congenita

PROJECT SUMMARY Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome. Mutations in NAP57 (aka dyskerin) account for about half of the genetically characterized cases of DC and constitute the severe X-linked form of DC (X-DC). NAP57 is the corner stone of all H/ACA ribonucleoproteins (RNPs), one of the two major classes of small nucleolar RNPs (snoRNPs). As such, NAP57 is essential for the stable expression of over 500 different H/ACA RNAs including human telomerase RNA (hTR). H/ACA RNPs are important for many basic cellular processes including ribosome biogenesis, regulation of protein synthesis, pre-mRNA splicing, and telomere maintenance. While it is well documented that DC impacts hTR and telomeres, it is controversial if and how X-DC mutations affect other H/ACA RNPs and their function. In unbiased fashion, this proposal examines the impact of DC on all H/ACA RNPs in three specific aims. First, we will characterize the H/ACA RNAome in general and in X-DC. We will exploit our specific antibodies against NAP57 to precipitate all associated H/ACA RNAs and identify and quantify them by next-generation sequencing (NAP57 RIPseq). Now, we will apply NAP57 RIPseq to determine the relative abundance of H/ACA RNAs in healthy carriers of X-DC and their affected sons. In addition to defining the complete cellular H/ACA RNA landscape, our studies will pinpoint the changes that occur in patient cells and underlie the disease phenotype. Second, we will define the role of Cajal bodies (CBs) in H/ACA RNP biogenesis and in X-DC. CBs are micron-sized nuclear bodies whose function, the maturation and modification of non-coding RNAs (e.g. hTR), is compromised in DC. We developed model cell lines using CRISPR/Cas 9 technology to knockdown Nopp140, a partner of NAP57, which results in the specific down regulation of NAP57 in CBs but not nucleoli, mimicking a deficiency observed in DC. We propose to analyze these cells using NAP57 RIPseq and common cell biological approaches to compare the results to those of DC patient cells. Third, we will visualize the structure of wild type and mutant NAP57 +/- SHQ1. Before association with an H/ACA RNA, NAP57 is complexed with the chaperone SHQ1. The over 50 DC mutations in NAP57 impair this interaction diminishing NAP57 in the cell. To visualize the molecular impact of DC mutations, we propose to build on our negative-stain electron microscopy (EM) of human full-length NAP57 to visualize by cryo-EM the structures of wild type and mutant NAP57 alone and in complex with SHQ1. In summary, our studies will inform on the molecular mechanism of DC and move the field forward by establishing the complete cellular H/ACA RNAome, shedding light on the function of CBs, and generating a first structure of full- length wild type and mutant NAP57. The latter will aid developing therapeutic approaches for DC, as well as for certain cancers, e.g. prostate, that are also characterized by mutations in NAP57 and SHQ1.