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.

Fig. 5.

Site-specific pseudouridylation of rRNA. (A) Nuclear extracts from targeted and control ES cell clones were incubated with labeled pseudouridylation templates. The sequence of ³²P-labeled oligo RNA substrates is shown. The asterisk indicates the positions of uridines that undergo pseudouridylation. Reactions were digested by RNase T2 and then separated by TLC. (B) The spots of uridine (Up) and Ψp were detected by autoradiography.

Fig. 1.

Targeting the Dkc1 locus in ES cells. (A) The targeting construct was a 6.5-kb EcoRI/HindIII fragment in which an IRES-Neo element had been cloned into a unique StuI site in the 3′ untranslated region. The positions of the probes are shown. The asterisk represents the position of the mutations A353V and G402E in exons 11 and 12, respectively. The positions of restriction enzyme sites used in confirmatory Southern blots (not shown) are indicated; B, BamHI; Bs, BspHI; H, HindIII; E, EcoRI. The Bs site in brackets is the site created with the A353V mutation. The probes were 5′, a single-copy 600-bp fragment from Dkc1 intron 9; IRES, a 200-bp HindIII/KpnI fragment from the IRES element; and 3′, a 500-bp BstX1/HindIII fragment from Mpp1 cDNA. (B) Northern hybridization. RNA was extracted from ES cells containing the wild-type Dkc1 gene with an IRES-NEO cassette in the 3′ untranslated region (WT+NEO) or ES cell clones targeted to contain point mutations as well as the IRES-NEO cassette (G402E and A353V). Twenty-five micrograms of total RNA was loaded in each lane and hybridized sequentially with probes for Dkc1 and β-actin.(C) Western blotting of nuclear extracts from ES cells. Ten micrograms of protein was loaded in each lane. Expression of dyskerin was detected with anti-dyskerin antiserum. TopoII-β was detected with a specific antibody as a loading control.

Fig. 2.

The effect of point mutations in Dkc1 on Terc RNA levels, telomerase activity, and telomere lengths. (A) Northern hybridization. RNA was extracted from ES cells containing the wild-type Dkc1 gene (WT+NEO) or ES cell clones targeted to contain point mutations (G402E and A353V). Twenty-five micrograms of total RNA was loaded in each lane and hybridized sequentially with probes for Terc and β-actin. (B) Telomerase activity in ES cells. Extracts in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) lysis buffer were prepared from Dkc1 mutant and control ES cell clones. The TRAP assay for telomerase activity was performed on 2.0 × 10^-2 (lane 1), 1.0 × 10^-2 (lane 2), and 0.5 × 10^-2 (lane 3) μg of protein. A total of 2.0 × 10^-2 μg of protein from heat-treated samples was used as negative control (lane 4). IC, internal positive control for PCR. (C) Telomere lengths in ES cells. Dkc1 mutant and control ES cell clones were passaged 60, 59, and 24 times. Telomeric terminal restriction fragments were analyzed by in-gel hybridization with a ³²P-end-labeled oligonucleotide (CCCTAA) probe. Lanes: 1, WT+NEO; 2, G402E; 3, A353V. The sizes of molecular mass markers are shown on the left.

Fig. 3.

The effect of Dkc1 mutations on H/ACA snoRNAs. Twenty micrograms of total RNA was loaded in each lane of a Northern blot, and the membrane was hybridized sequentially with end-labeled oligonucleotide probes specific for the murine H/ACA snoRNA species indicated. The C/D box snoRNA U14 was used as a loading control.

Fig. 4.

Pseudouridylation in 18S or 28S rRNA from G402E and A353V ES cells. 18S and 28S rRNA of Dkc1 mutant and control ES cells was labeled with ³²P-labeled orthophosphate, extracted, and gel-purified. After digestion with RNase T2, each sample was separated by two-dimensional TLC. The positions of the labeled ribonucleotides are indicated. From each TLC plate, spots of uridine (Up) and pseudouridine (Ψp) were excised and quantitated by Cerenkov counting. The ratio between incorporation into uridine and Ψp was calculated and is shown on the histogram where the WT+NEO, G402E, and A353V samples are compared with the WT+NEO sample (100%).

Fig. 6.

The effect of Dkc1 mutations on rRNA processing. (A) rRNA was labeled with 5 μCi/ml ³H-labeled uridine for 30 min, then incubated in nonradioactive media for 2 h. In both experiments, RNA was extracted, loaded according to cell number (1.0 × 10⁶ cells), and separated through 1% agarose formaldehyde gel. (B) Pulse–chase labeling experiment. Cells were labeled with 50 μCi/ml ³H-labeled methyl methionine for 30 min and chased in cold methionine for 15, 30, 45, and 60 min. Lanes: 1, WT+NEO; 2, G402E; 3, A353V. The sizes of the processing intermediates and the mature RNA species are indicated at the left.