Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance

Abstract

POT1 is a single-copy gene in yeast and humans that encodes a single-strand telomere binding protein required for chromosome end protection and telomere length regulation. In contrast, Arabidopsis harbors multiple, divergent POT-like genes that bear signature N-terminal OB-fold motifs, but otherwise share limited sequence similarity. Here, we report that plants null for AtPOT1 show no telomere deprotection phenotype, but rather exhibit progressive loss of telomeric DNA. Genetic analysis indicates that AtPOT1 acts in the same pathway as telomerase. In vitro levels of telomerase activity in pot1 mutants are significantly reduced and are more variable than wild-type. Consistent with this observation, AtPOT1 physically associates with active telomerase particles. Although low levels of AtPOT1 can be detected at telomeres in unsynchronized cells and in cells arrested in G2, AtPOT1 binding is significantly enhanced during S-phase, when telomerase is thought to act at telomeres. Our findings indicate that AtPOT1 is a novel accessory factor for telomerase required for positive telomere length regulation, and they underscore the coordinate and extraordinarily rapid evolution of telomere proteins and the telomerase enzyme.

Figure 1

Telomere phenotypes in AtPOT1-deficient Arabidopsis. (A) Genomic map and coding region of the AtPOT1 locus. Rectangles are exons; black lines represent introns. The position of T-DNA insertions in the pot1-1 and pot1-2 alleles are shown. OB1 and OB2 indicate two predicted OB-folds in the AtPOT1 protein. The position of the peptide used to raise P1-P1 and P1-P2 antibodies is indicated. Both peptides were raised against a similar AtPOT1 region, but P1-P2 peptide is slightly longer (see Materials and Methods). (B) RT–PCR analysis of the AtPOT1 gene expression in pot1-1 and pot1-2 mutants. Primer pairs 1–2 and 1–3 (shown as arrowheads in panel A) were used to analyze gene expression. (C) IP of recombinant ³⁵S-labeled AtPOT1 protein. IP efficiencies for P1-P1 and P1-R antibodies are indicated. (D) Detection of endogenous AtPOT1 protein in wild-type and pot1-1 callus. AtPOT1 was immunoprecipitated and detected by Western blot analysis using P1-P2 antibody. The arrow indicates the 55 kDa endogenous AtPOT1 protein immunoprecipitated from wild-type callus. The asterisk indicates a nonspecific cross-reacting protein. (E) TRF analysis of DNA from six siblings segregating from a heterozygous pot1-1 parent. (F) TRF analysis of a pot1-2 mutant. (G) Multi-generational TRF analysis of pot1-1. DNA samples from two individual pot1-1 plants from the second (G2), fourth (G4), and sixth (G6) generation of self-pollination were analyzed. Blots shown in panels E, F and G were hybridized with a radiolabeled telomeric DNA probe. Molecular weight markers are indicated. Plants analyzed in panel G are from the WS ecotype, plants in panel E are from a Columbia-WS cross, and plants in panel F are from Columbia. Telomeres in wild-type WS plants are typically longer than those in Columbia (Shakirov and Shippen, 2004).

Figure 2

Parent–progeny analysis reveals the same rate of telomere shortening in pot1-1, pot1-2, and tert mutants. (A, B) TRF analysis of bulk telomeric DNA from pot1-1 and pot1-2 parents (P) and two progeny (1 and 2) using a telomeric probe. (C) Subtelomeric TRF analysis of DNA from a pot1-1 parent and two progeny. DNA blots were hybridized with a probe corresponding to unique subtelomeric regions on 2R and 1L chromosome arms. The asterisk indicates a cross-hybridizing band. (D) PETRA analysis of the 2R telomere in a pot1-1 parent and two progeny. (E) PETRA analysis of the 2R and 5R telomeres in a parent homozygous for tert and heterozygous for pot1-1 (ttPp), and its tert (ttPP) (a) and pot1-1 tert (ttpp) (b) progeny. The two PETRA bands detected in the 2R reaction may represent different size telomeres on homologous chromosomes or two populations of cells (Shakirov and Shippen, 2004). A telomeric probe was used to detect PETRA products.

Figure 3

AtPOT1 functions in the telomerase pathway. (A) TRF analysis of pot1-1 tert mutants. Results for eight progeny (two for each genotype) that were segregated from a parent heterozygous for pot1-1 and tert are shown. (B) TRF analysis of telomeres in pot1-1 tert parent (P) and its two progeny (1 and 2) are shown. (C) TRF analysis of pot1-1 ku70 mutants. Results for progeny segregated from a parent heterozygous for pot1-1 and ku70 are shown. Two different progeny were analyzed for each genotype. The blot was hybridized with a telomeric DNA probe.

Figure 4

AtPOT1 interacts with the telomerase RNP. (A) TRAP assay results for wild-type (WT), pot1-1, and pot1-2 flowers. Results for extracts prepared from 10 different plants are shown in lanes 3–12. (B) TRAP assay results for wild-type and pot1-2 mutant flowers. 10 × and 100 × dilutions of protein extracts were used for TRAP as indicated. In panels A and B, extract prepared from Arabidopsis suspension culture (lane 1) served as the positive control (+). (C) TRAP assay following AtPOT1 IP from suspension culture using P1-P1 or P1-R antibodies. (D) TRAP assays with P1-P2 immunoprecipitates from wild-type and 4-day-old pot1-2 seedlings extracts. (E) TRAP assay results for P1-P1 antibody immunoprecipitates. IP from suspension culture extract was performed with no addition of peptide (−), 100 × excess of P1-P1 peptide (P1), or 100 × excess of a nonspecific AtPOT2 peptide (P2). (F) TRAP assays with eluates from P1-R IP in the presence of NaCl. NaCl concentrations are indicated. (G) TRAP assays with P1-R immunoprecipitates from unsynchronized suspension culture (unsyn.) or S-phase and G2-phase synchronized cells. The relative amount of active telomerase precipitated in the reaction is indicated. In all panels, IP with no antibody added ((−) IP), or with preimmune serum (Preim. IP), was used as a negative control.

Figure 5

AtPOT1 is associated with telomeric chromatin in S-phase. (A) FACS analysis of Arabidopsis suspension culture cells synchronized with aphidicolin. Data are shown for unsynchronized (US) and synchronized cells at 0, 3, 6, and 9 h after release from aphidicolin arrest. (B) Example of ChIP analysis on synchronized suspension culture extracts using P1-P2 antibody or preimmune serum. Immunoprecipitated DNA was monitored on slot blot using a radiolabeled telomeric or rDNA probe. (C) Quantitation of AtPOT1 association with telomeric DNA. The average of results from four independent experiments is shown. The solid black line indicates the ratio of the telomeric DNA signal obtained with the POT1 antibody relative to the preimmune sera control. As a negative control, the rDNA signal obtained with the POT1 antibody relative to the preimmune sera (gray dashed line) is shown. (D) Western blot analysis of AtPOT1 protein. Extracts from synchronized cells were precipitated with P1-P2 antibody, followed by P1-P2 Western blot analysis. Commassie-stained inputs (right) are shown as loading controls.