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First published online 2 October 2007
doi: 10.1242/jcs.004119

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Arabidopsis POT1A interacts with TERT-V(I8), an N-terminal splicing variant of telomerase

Department of Cellular and Developmental Biology, John Innes Centre, Norwich, NR4 7UH, UK

View larger version (22K): [in this window] [in a new window]   Fig. 1. Schematic of the pDEST-GADT7 and pDEST-GBKT7 expression cassettes. The Gateway cassette [composed of the recombination sites attR1 and attR2, the chloramphenicol resistance gene (CmR) and the ccdB gene] was inserted into the SmaI site of the yeast two-hybrid vectors pGADT7 and pGBKT7. LR recombination with the target gene replaces the region between the attR1 and attR2 recombination sites to create in-frame fusions with either the GAL4 activation domain (GAL4 AD) fused to the HA-tag in the case of pDEST-GADT7 or the GAL4 binding domain (GAL4 BD) fused to the c-Myc tag for pDEST-GBKT7. The promoter from ADH1 (PADH1) and the ADH1 terminator (TADH1) were used to control the expression of the gene fusion in yeast, and the T7 promoter (PT7) was used for in vitro expression. Nucleic acid sequence and predicted amino acid sequence of the junctions between the original yeast two-hybrid vectors and the gateway cassette are given below each vector.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Identification of POT1A-interacting proteins from the yeast two-hybrid screen. Growth tests of diploid yeast strains on SD-LW (non-selective) and SD-LWHA (selective) media. The activation domain (AD)-fusion protein genotypes of diploids are given on the left of the panel, whereas the binding domain (BD)-fusion protein genotypes (top) are as follows: 1, TERT; 2, CIPK21; 3, At1g29040; and 4, At5g67385.

View larger version (20K): [in this window] [in a new window]   Fig. 3. POT1A interacts with TERT-V(I8) in vitro. The presence (+) or absence (–) of particular components is indicated above: myc, translation mix using control vector, pGBKT7 as template; myc-POT1A, translation mix using POT1A cloned in pDEST-GBKT7; and [35S-met]-HA-TERT-V(I8), translation mix using TERT-V(I8) cloned in pDEST-GADT7. The upper panel [labelled AtTERT V(I8)] is an autoradiograph revealing the presence or absence of the TERT fusion protein. The lower panel (labelled POT1A) is a western blot revealing the presence or absence of the POT1A fusion protein. Heavy chain IgGs are indicated by arrowheads. Lane 1, radiolabelled [35S-met]-TERT-V(I8) synthesis reaction; lane 2, POT1A synthesis reaction (myc-tagged); lanes 3 and 4, input: the mixed proteins after an overnight incubation with antibodies and protein A beads, correspond to 0.2% of the total amount of protein used for immunoprecipitation; lanes 5 and 6, IP anti-myc: the proteins remaining bound to the protein A beads after immunoprecipitation.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Mapping of the interaction between POT1A and TERT. (A) Structure of the Arabidopsis TERT protein, variants and truncations. Motifs in TERT (GQ, CP, QFP, T, 1, 2, A, B, C, D and E) were located according to Fitzgerald et al. (Fitzgerald et al., 1999) and Xia et al. (Xia et al., 2000). Putative nuclear localisation signals (NLS) are shown as triangles. The prediction of the peptide encoded by the TERT-V(I8) mRNA variant is shown and the stop codon is marked (*). Truncation constructs of TERT are shown below and correspond to amino acids 1-323 (TERT1-323); 1-413 (TERT1-413); and 414-1123 (TERT414-1123). (B) Quantification of the interaction between POT1A [as pBD fusion (i) or as pAD fusion (ii)] and various telomerase peptides [shown in the schematic from top to bottom: TERT-V(I8), TERT1-323, TERT1-413, TERT414-1123, TERT, pGADT7 (control empty vector)]. The central panel shows serial dilutions of diploid cells on non-selective (SD-LW), low-selective (SD-LWH) and high-selective (SD-LWHA) media. The panel on the right-hand side shows the results of a -galactosidase liquid culture assay, in Miller units, obtained from two separate experiments, each containing three replicates. Bars represent the standard errors. (C) Western blot analysis of protein expression in diploid cells. The genotype of the cells is indicated above the blots. Lanes 1-3 and 7-9 were probed with anti-HA antibodies; lanes 4-6 and 10-12 were probed with anti-myc antibodies. Arrowheads indicate the positions of expected molecular masses for the fusions tested.

View larger version (34K): [in this window] [in a new window]   Fig. 5. Alternative splicing of the Arabidopsis TERT gene. (A) Schematic indicating the predicted intron/exons in the full-length TERT gene and those of the TERT-V(I8) variant. Size of the transcripts is given in bp. Exons are represented as black boxes and introns as striped boxes. Scale (bp) is given in the upper right corner. Numbered arrows show the location of the primers used in the RT-PCR experiments (see B). (B) RT-PCR analysis of the 5' end of TERT transcripts from inflorescences and leaves using the sets of primers as shown below the TERT gene in A. Black arrowheads indicate amplification of unspliced transcripts, and white arrowheads indicate spliced and alternatively spliced variants. (C) Schematic representation of the alternative splicing transcript (AST) sequences obtained after RT-PCR using the different primers sets as shown for each AST (ASTa-ASTg). Ratios given in brackets represent the frequency of each variant after sequencing. For primer sets 1-2 and 1-6, the remaining clones sequenced corresponded to unspliced mRNA. Sizes of each variant are given in bp. Retained introns are represented as striped boxes and the partially deleted exon 5 is shown as a white box.

View larger version (47K): [in this window] [in a new window]   Fig. 6. Localisation of the Arabidopsis POT1 proteins. (A) POT1A, POT1B and KU80 localise both to the cytoplasm and to the nucleus of Arabidopsis Col-0 cells. Top panel: localisation of telomeric POT1A, POT1B and KU80 fused to GFP in living cells. Bottom panel: Arabidopsis Col-0 cells transiently transformed with POT1A, POT1B and KU80 fused to GFP were extracted with detergent. Localisation of the proteins is shown in the GFP panel. Arrows indicate nuclei. The chromatin was stained with DAPI (DAPI panel) and co-localisation of the signals obtained with GFP and DAPI are shown in the merge panel. (B) Nuclear localisation of TERT-V(I8) and of a TERT truncation fused to GFP in Arabidopsis Col-0 cells. The two GFP fusions label the nucleoli (arrows) and the entire nuclei in some cells. Bars, 4 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Conceptual models for POT1-TERT interaction in telomere synthesis. (A) Alternatively spliced TERT, TERT-V(I8) [V(I8)], acts to displace POT1A bound to telomeres, and this protein interaction allows TERT to be recruited to telomeres, where it can synthesise DNA repeats. (B) The GQ domain of TERT is hidden within the protein structure and is inaccessible to POT1A interaction. Following signals or modifications that trigger conformational changes of the TERT protein, the GQ domain of TERT become accessible to POT1A and the interaction mediates the recruitment of TERT to telomeres.

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