Mouse dyskerin mutations affect accumulation of telomerase RNA and small nucleolar RNA, telomerase activity, and ribosomal RNA processing

Abstract

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.

How a single protein complex accommodates many different H/ACA RNAs

“…Do mutations in NAP57, therefore, specifically affect its interaction with telomerase RNA but not with the other one hundred or so H/ACA RNAs? The answer appears to be no as the DC phenotype can be reproduced by mutations in NAP57 that, in the absence of telomere defects, impair ribosome biogenesis (and possibly pre-mRNA splicing) through reduced RNA pseudouridylation [24,25]. Crystal structure of the H/ACA protein complex of Gar1 (blue), Nop10 (red), and Cbf5 with its catalytic domain (green), PUA domain (cyan), and amino terminus (yellow).…”

Telomerase and its potential for therapeutic intervention

“…Telomere binding proteins include but are not limited to the telomeric repeat binding factors 1 and 2 (TRF1 and TRF2), TRF-1 interacting protein 2 (TIN2), POT1 (protection of telomeres 1), as well as tankyrase (TRF1-inactivating ankyrin-related adenosine diphosphate (ADP)-ribose polymerase), a member of the PARP family proteins ( Figure 2, Table 1) (van Steensel and de Lange, 1997;Smith et al, 1998;Karlseder et al, 1999). Dyskerin is a hTERC associated protein that has a structural function (Mochizuki et al, 2004).…”

A pathogenic dyskerin mutation impairs proliferation and activates a DNA damage response independent of telomere length in mice

“…Moreover, a universal feature of all forms of DC is the presence in the patients of very short telomeres (20). However, the X-linked form of DC is usually more severe, and some defects in ribosome biogenesis have been observed in mice and in mouse embryonic stem cells with targeted dyskerin mutations (14,15) suggesting that functions of dyskerin other than the prevention of short telomeres may contribute to the disease.…”

“…The mutations identified in DC patients therefore only impair but do not destroy dyskerin function. To what extent each of the different dyskerin functions is affected and contributes to the pathogenesis of disease is controversial (14)(15)(16), although the finding of mutations in genes encoding other telomerase and telomere components in DC (17)(18)(19) and studies in DC cell lines showing impaired telomere maintenance (11,16) emphasize the importance of the telomere maintenance pathway in disease pathology. Moreover, a universal feature of all forms of DC is the presence in the patients of very short telomeres (20).…”