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The accumulation and not the specific activity of telomerase ribonucleoprotein determines telomere maintenance deficiency in X-linked dyskeratosis congenita.

ABSTRACT: X-linked dyskeratosis congenita (X-DC) is caused by mutations in the housekeeping nucleolar protein dyskerin. Amino acid changes associated with X-DC are remarkably heterogeneous. Peripheral mononuclear blood cells and fibroblasts isolated from X-DC patients harbor lower steady-state telomerase RNA (TER) levels and shorter telomeres than healthy age-matched controls. Previously, we showed that retroviral expression of recombinant TER, together with expression of recombinant telomerase reverse transcriptase, restored telomere maintenance and proliferative capacity in X-DC patient cells. Using rare X-DC isoforms (ΔL37 and A386T dyskerin), we showed that telomere maintenance defects observed in X-DC are solely due to decreased steady-state levels of TER. Disease-associated reductions in steady-state TER levels cause deficiencies in telomere maintenance. Here, we confirm these findings in other primary X-DC patient cell lines coding for the most common (A353V dyskerin) and more clinically severe (K314R and A353V dyskerin) X-DC isoforms. Using cell lines derived from these patients, we also examined the steady-state levels of other hinge-ACA motif RNAs and did not find differences in their in vivo accumulations. We show, for the first time, that purified telomerase holoenzyme complexes from different X-DC cells have normal catalytic activity. Our data confirm that dyskerin promotes TER stability in vivo, endorsing the development of TER supplementation strategies for the treatment of X-DC.

SUBMITTER: Zeng XL

PROVIDER: S-EPMC3263989 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ?L37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ?L37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human patients have so far not shown defects in pseudouridylation or ribosomal RNA processing. None of the mutant iPS cells presented here show decreased pseudouridine levels in rRNA or defective rRNA processing suggesting telomere maintenance defects account for most of the phenotype of X-linked DC. Finally gene expression analysis of the iPS cells shows that WNT signaling is significantly decreased in all mutant cells, raising the possibility that defective WNT signaling may contribute to disease pathogenesis.

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The accumulation and not the specific activity of telomerase ribonucleoprotein determines telomere maintenance deficiency in X-linked dyskeratosis congenita.

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