Dyskerin is a component of the Arabidopsis telomerase RNP required for telomere maintenance

Abstract

Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.

FIG. 1.

Expression and localization of AtNAP57. (A) RT-PCR analysis of the AtNAP57 transcript in different plant tissues. AtTRP1H encodes a putative double-strand telomere binding protein (23) and was used as a loading control. (B) Recombinant AtNAP57 and AtTERT proteins were expressed in RRL and labeled with [³⁵S]methionine (*). Proteins were immunoprecipitated with an antibody (Ab) raised against mouse dyskerin (α-dyskerin) or an antibody raised against an N-terminal peptide in AtTERT (α-TERT). Relevant lanes are shown. (C) Immunolocalization of AtNAP57 and AtTERT in Arabidopsis suspension culture cells and in floral buds. Nuclei were stained with DAPI or the antibodies discussed above.

FIG. 2.

AtNAP57 associates with Arabidopsis telomerase RNP. (A) Western blot analysis with FLAG antibody on plant extracts from the wild type (WT) or transformants bearing FLAG-tagged AtNAP57 (FN). (B) Western blot analysis of input or immunoprecipitates (IP) obtained with FLAG antibody on extracts from the WT and FLAG-tagged AtNAP57 transformants. Four percent of input and 30% of IP was loaded on the gel. (C) TRAP assay results for WT and FN extracts before (input) or after immunoprecipitation (IP) with FLAG antibody. Immunoprecipitates were assayed in duplicate. (D) Top panel, TRAP assay results for cell culture extracts immunoprecipitated with preimmune serum (PI) and anti-TERT (α-TERT) peptide antibody. The sample shown in the far-right lane was pretreated with 100 μg/ml of RNase A prior to immunoprecipitation. Bottom panel, α-TERT immunoprecipitates were subjected to Western blot analysis using the dyskerin antibody (α-dyskerin). Fifteen percent of input and 60% of IP was loaded on the gel.

FIG. 3.

AtNAP57 weakly associates with AtPOT1a. (A) Coimmunoprecipitation experiments were performed with the full-length recombinant AtKU70, AtTERT, and AtPOT1a proteins, labeled using [³⁵S]methionine (*), and T7-tagged AtKU80, AtKU70, and AtNAP57. Proteins were incubated with either T7 antibody (Ab) beads (control) or T7 beads and the indicated T7-tagged unlabeled proteins. The supernatant (S) and pellet (P) fractions were loaded in equal amounts. (B) Results of yeast two-hybrid analysis are shown. The indicated yeast crosses were performed and plated on medium lacking leucine, tryptophan, and histidine. Results of a colony lift β-galactosidase (β-gal) assay are shown. The blue color is indicative of protein interaction.

FIG. 4.

AtNAP57 is an essential gene in Arabidopsis. (A) Schematic diagram of the AtNAP57 coding region showing the position of the T-DNA insertion, pseudouridine synthase domain (TruB), pseudouridine synthase, archeosine transglycosylase domain (PUA), and nuclear localization signal (NLS). (B) Siliques (seed pods) from wild-type (WT) or nap57^+/− plants were visualized by microscopy. A reduced seed set was observed for nap57^+/− plants, implying that the homozygous mutation is lethal. (C) TRF analysis of WT, first-generation (G1), or second-generation (G2) nap57^+/− plants. Molecular size markers are indicated.

FIG. 5.

The T66A mutation in AtNAP57 results in the establishment of a shorter telomere length set point. (A) Sequence alignment of human dyskerin and AtNAP57 proteins. Conserved residues are highlighted in gray boxes, and the threonine residue targeted for mutagenesis is denoted by an asterisk. (B) Overview of the process for introduction of the T66A mutation in AtNAP57 into nap57^+/− plants. (C) TRF analysis of first, second, and third (T1, T2, and T3) generations of T66A transformants. The T1 plant whose telomeres were analyzed in the left panel, lane 4, was used as the parent for T2 progeny plants analyzed in the middle panel. The T2 plant represented in the middle panel, lane 7, was the parent for the T3 progeny analyzed in the right panel. DNA samples were not run as far into the gel shown the right panel as in the other two gels. (D) Graphic representation of bulk telomere length size range and peak telomere length (indicated by -) for WT and T66A transformants is shown. Arrows indicate telomere length measurements for plants used as T1 and T2 parents.

FIG. 6.

The T66A mutation in AtNAP57 affects telomere length regulation on individual chromosome ends and decreases telomerase activity in vitro. (A) PETRA results are shown for the WT and individual nap57^+/−, T1, T2, and T3 T66A nap57 transformants with short telomeres (S) and a T3 T66A nap57 transformant with wild-type-length telomeres (L). The telomeres monitored are indicated. 2R, right arm of chromosome 2; 3L, left arm of chromosome 3; 4R, right arm of chromosome 4. (B) Graphic representation of PETRA products obtained in each reaction as determined by visual inspection. (C) TRAP assay results for the WT and nap57^+/− and T66A nap57 transformants. Reactions were conducted using 1:50, 1:500, and 1:5,000 dilutions of protein extracts. (D) Results of real-time TRAP. The top panel shows raw data for three (each) of the WT and nap57^+/− and T66A nap57 transformants. The dashed line represents the threshold cycle for TRAP product detection. The bottom panel shows a histogram of the telomerase activity levels for nap57^+/− and T66A transformants relative to those for the WT. Extracts from 10 individual plants from each genotype were monitored.

FIG. 7.

The T66A mutation in AtNAP57 reduces telomerase activity in vivo. (A) Schematic diagram of genetic crossing scheme to generate ku70^−/− mutants carrying the T66A nap57 allele. (B) TRF analysis of seven ku70^−/− T66A nap57 plants and one ku70^−/− nap57^+/− control plant is shown.