The Importance of DKC1 Pseudouridine Capacity in Translational Control and X-linked Dyskeratosis Congenita Pathogenesis

X-linked Dyskeratosis Congenita (X-DC), which is the most severe form of the hematopoetic disorder DC, is characterized by mutations in the DKC1 gene which codes for dyskerin. We have previously found that dyskerin catalyzes the conversion of uridine to pseudouridine (Ψ) of ribosomal RNA (rRNA) and plays an important role in the translational control of a specific subset of messenger RNAs (mRNAs) that share a common feature: an internal ribosome entry site (IRES) in their 5’ untranslated region. Dyskerin assembles with small nucleolar RNAs (snoRNAs) that guide this protein to the site of rRNA modification, and mutations in dyskerin result in a decrease in levels of snoRNAs. Dyskerin also has additional functions in telomere maintenance and splicing activity. However, we hypothesize that the translational defects resulting from the reduced pseudouridylation capacity of dyskerin is the primary contributor to the disease. Site-directed mutagenesis was employed to introduce a point mutation into the catalytic domain of DKC1 that is responsible for pseudouridylation. Both wild type DKC1 and the mutant DKC1 genes were inserted into a lentiviral plasmid through cloning. These constructs were then used to infect both X-DC patient and normal lymphoblasts to generate stable cell lines. We analyzed the levels of snoRNAs and IRES-dependent translation in these lymphoblasts. We also employed a novel technique to measure the global rRNA pseudoridylation levels in these cell lines. These studies will allow us to distinguish the major role dyskerin plays in translational control and how it contributes to X-DC pathogenesis.