The chimera reconstitutes human telomerase activity upon hTERT expression in vitro. (A) Upper panel. The chimera reconstitutes telomerase activity by TRAP. Telomerase complexes were reconstituted in RRL, and telomerase activity was detected using TRAP. IC indicates the internal PCR control. Lower panel. In vitro synthesis of hTERT and tTERT. 1 µl of the corresponding RRL reaction was analyzed by SDS–PAGE. (B) Upper panel. tTR does not function with hTERT to reconstitute telomerase activity by TRAP. Telomerase complexes were reconstituted in RRL, and telomerase activity was detected by TRAP, using a modified primer complementary to the template sequence of tTR (ACT-C₄A₂) (lanes 1–9) or the ACX primer (lanes 10 and 11). Lower panel. In vitro synthesis of hTERT and tTERT. The corresponding RRL reaction of 1 µl was analyzed by SDS–PAGE. (C) hTR1-56 and hTR160-451 do not reconstitute telomerase activity as measured by TRAP. The same reconstitution method as in (A) was used. (D) A mutation within the tTR pseudoknot sequences in htTR significantly reduces the activity reconstituted by the chimeric enzyme. The same method as in (A) has been used. %WT indicates the average percentage of WT telomerase activity derived by quantification of the variants' telomerase activities relative to the WT telomerase activity and the standard deviation. A total of five experiments were performed. (E) Diagram depicting the predicted alignments of the oligonucleotides (used in F and H) with the hTR template. The arrow indicates the translocation of the primer along the template after the first round of DNA synthesis by the telomerase enzyme. P stands for primer length. (F) The chimeric enzyme is nonprocessive. RRL-reconstituted human telomerase catalytic activity was tested using the direct primer extension telomerase assay with the indicated 3′ end-labeled biotinylated primers. (P + 4), (P + 4) + 6, (P + 6), (P + 6) + 6 indicate the position of the products synthesized by telomerase (arrows, asterisks). (G) The chimeric enzyme nonprocessively elongates a Tetrahymena-like telomeric oligonucleotide, (T₂G₄)₃. The same method as in (F) was used. (H) The htTR stem IIIa- and stem IIIb-reconstituted enzymes are nonprocessive. The same method as in (F) was used.

(A and B) The chimera dimerizes and alterations of the stem IIIa and stem IIIb in the Tetrahymena pseudoknot structure do not inhibit its dimerization. Representative results of dimerization for htTR mutants are shown. M and D correspond to monomer and dimer positions, respectively. (B) Quantification of the dimerization results for the htTR mutants. A minimum of three independent experiments was performed for each RNA. (C and D) Mutation in the J7b/8a region of the chimera affected its dimerization. (C) Representative results of dimerization for htTR and hTR mutants are shown. (D) Quantification of the dimerization values for the hTR and htTR mutants. Average dimerization values did significantly differ from hTR (one asterisk) or the chimera (two asterisks) in a Student's t-test.

(A and B) Secondary structures of hTR and tTR [adapted from (10,13)]. Helices I, II, III, IIIa/IIIb and IV and the template are indicated for tTR. The template, helices P1, P2a.1, P2a, P2b, P3 (CR2–CR3), P6.1, the H/ACA box, the CR4–CR5 and J7b/8a regions are indicated for hTR. The arrows indicate the boundaries of the mutations in the tTR and hTR variants used in this study. The boxes highlight the hTR and tTR nucleotides deleted and inserted respectively, to construct the htTR chimera.

The chimera interacts with hTERT in vitro. (A) ³⁵S-labeled hTERT was synthesized in RRL in the presence of equal amounts of ³²P-labeled hTR and increasing amounts of unlabeled competitor RNAs, hTR or htTR (3, 30 and 300 ng). The formed ribonucleoprotein complexes were immunoprecipitated with an anti-hTERT antibody and visualized by SDS–PAGE. No competition (NC) (B) Quantification of the association between hTR and hTERT in the presence of competitor RNAs, expressed relative to the NC control. At least three independent experiments were performed. (C). The chimera interacts with hTERT RID2. The same method as in (A) was used. (D) Quantification of the association between hTR and hTERT RID2 in the presence of competitor RNAs.

The chimera is defective in telomere elongation. (A) Telomerase activity of the VA13-hTERT clones. A total of 1 µg of whole-cell extracts was assayed for telomerase activity by TRAP. As a positive control, 1 µg of whole-cell extract from telomerase-positive HL60 was tested for telomerase activity by TRAP, IC, internal PCR control. (B) hTR and htTR expression in the VA13-hTERT clones. cDNA from VA13-hTERT clones transfected with pcDNA3-hTR, pcDNA3-htTR or pcDNA3 were analyzed by PCR for the expression of hTR or htTR. The plasmids phTR + 1 and phtTR were used as positive controls for the PCR and GADPH as an IC for the integrity of the cDNAs. (C) hTERT expression in the VA13-hTERT clones. cDNA from VA13-hTERT clones transfected with hTR, htTR or the vector were analyzed for the expression of hTERT by PCR. (D) Quantification of the naked extremities in VA13-hTERT clones by Q-FISH. A minimum of 15 metaphases were prepared for each clone (as indicated by the number on the bar of the graph). n.s., non statistically significant; m, number of metaphases prepared. The results were expressed as a ratio of naked extremities over the total extremities measured by Q-FISH in one vector clone, two clones expressing hTR and in three clones expressing htTR. Average values were analyzed by a Student's t-test, and P-values from comparison between different clones are indicated. (E and F) The chimera assembles with hTERT in vivo but is not active by TRAP. Telomerase was immunoprecipitated from whole-cell extracts made from hTR-transfected, htTR-transfected or vector-transfected clones and the telomerase-positive human cell line, HL60, using an anti-hTERT antibody (E) 10% of the resulting beads were used to perform a TRAP assay on the immunopurified complex. Thirty percent of the beads were used to reveal the presence of the hTERT protein by western blot analysis with the same antibody used for the immunopurification (F, top panel). Sixty percent of the beads were used to reveal the presence of hTR and htTR in the immunopurified complex by northern blot with a probe directed against a common region of the two RNAs (F, bottom panel).