Dyskerin is a nucleolar protein present in small nucleolar ribonucleoprotein particles that modify specific uridine residues of rRNA by converting them to pseudouridine. Dyskerin is also a component of the telomerase complex. Point mutations in the human gene encoding dyskerin cause the skin and bone marrow failure syndrome dyskeratosis congenita (DC). To test the extent to which disruption of pseudouridylation or telomerase activity may contribute to the pathogenesis of DC, we introduced two dyskerin mutations into murine embryonic stem cells. The A353V mutation is the most frequent mutation in patients with X-linked DC, whereas the G402E mutation was identified in a single family. The A353V, but not the G402E, mutation led to severe destabilization of telomerase RNA, a reduction in telomerase activity, and a significant continuous loss of telomere length with increasing numbers of cell divisions during in vitro culture. Both mutations caused a defect in overall pseudouridylation and a small but detectable decrease in the rate of pre-rRNA processing. In addition, both mutant embryonic stem cell lines showed a decrease in the accumulation of a subset of H/ACA small nucleolar RNAs, correlating with a significant decrease in site-specific pseudouridylation efficiency. Interestingly, the H/ACA snoRNAs decreased in the G402E mutant cell line differed from those affected in A353V mutant cells. Hence, our findings show that point mutations in dyskerin may affect both the telomerase and pseudouridylation pathways and the extent to which these functions are altered can vary for different mutations.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome associated with increased cancer susceptibility. The X-linked form is due to mutations in the DKC1 gene encoding dyskerin, a nucleolar protein predicted to be involved in rRNA processing and associated with the telomerase complex. Available evidence suggests the pathology of DC is due to telomerase defects. We have used the inducible Cre/ loxP system to produce deletions in the murine Dkc1 gene in early embryogenesis. A large deletion lacking exons 12 -15 and a small deletion lacking only the last exon, were produced. We found both deletions showed a parent-of-origin effect with 100% embryonic lethality when the mutation occurred on the maternal Dkc1. Embryonic analysis at day E7.5 and E9.5 showed no male embryos carrying either deletion whereas females with maternally derived deletions died around day E9.5, with degeneration of the extra embryonic tissue, in which the paternal X-chromosome is inactivated. Female mice carrying the deletion in the paternally derived Dkc1 show extreme skewing of X-inactivation with the wild type Xchromosome active in all cells. Since mice with no telomerase are viable in the first generations the lethality we observe is unlikely to be due to the effects of mutated dyskerin on telomerase activity.

Dyskerin is a component of small nucleolar ribonucleoprotein complexes and acts as a pseudouridine synthase to modify newly synthesized ribosomal, spliceosomal, and possibly other RNAs. It is encoded by the DKC1 gene, the gene mutated in X-linked dyskeratosis congenita, and is also part of the telomerase complex. The yeast ortholog, Cbf5, is an essential protein, but in mammals the effect of dyskerin ablation at the cellular level is not known. Here we show that mouse hepatocytes can survive after induction of a Dkc1 deletion. In the absence of dyskerin, rRNA processing is inhibited with the accumulation of large precursors, and fibrillarin does not accumulate in nucleoli. A low rate of apoptosis is induced in the hepatocytes, which show an induction of the p53-dependent cell cycle checkpoint pathway. Signs of liver damage including an increase in serum alanine aminotransferase activity and a disordered structure at the histological and macroscopic levels are observed. In response to carbon tetrachloride administration, when wild-type hepatocytes mount a rapid proliferative response, those without dyskerin do not divide. We conclude that hepatocytes can survive without dyskerin but that the role of dyskerin in RNA modification is essential for cellular proliferation.

To investigate the needs of parents of premature infants hospitalized in a neonatal intensive care unit (NICU) and explore associated factors. This study includes a convenience sample of 198 parents of premature infants hospitalized in a NICU in Chenzhou, Hunan province, China. Parents completed a study-specific questionnaire and the Chinese version of Critical Care Family Need Inventory (CCFNI). Analysis was conducted by multiple linear regression. Parents rated 30 of 45 items on the Chinese version of the CCFNI as important or very important. Items on the assurance, information, and proximity subscales were perceived as the most important, while items on the comfort subscale were the least important. Gender of parents and experience of visiting a NICU were significantly associated with the total needs of the parents. These insights should be used to inform clinical practice in the NICU.

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