The reverse transcriptase component of the Tetrahymena telomerase ribonucleoprotein complex

Abstract

Telomerase is a eukaryotic reverse transcriptase that adds simple sequence repeats to chromosome ends by copying a template sequence within the RNA component of the enzyme. We describe here the identification of a Tetrahymena telomerase protein with reverse transcriptase motifs, p133. This subunit is associated with the previously identified Tetrahymena telomerase RNA and the telomerase proteins p80 and p95 in immunoprecipitation assays. Therefore, all four known Tetrahymena telomerase components are present in a single complex. Expressed in rabbit reticulocyte lysate, recombinant p133 and telomerase RNA alone catalyze a reverse transcriptase activity with some similarities to and some differences from native Tetrahymena telomerase. These experiments suggest a complexity of telomerase structure and function.

Figure 1

The cDNA and genomic region encoding p133. (A) The cDNA sequence of p133 is indicated as an open box. Locations of the predicted start codon (ATG) and stop codon (TGA) are shown by thick lines. Locations of the 18 introns removed from the cDNA are shown by thin lines. Conserved sequence motifs are lettered and numbered. (B) The motif region sequence of Tetrahymena thermophila p133 (Tt133, Top) and Euplotes aediculatus p123 (Ea123, Bottom; ref. 10) are aligned over the indicated amino acids. Motif designations are given above the alignment, and the previously derived consensus (19) is given below.

Figure 2

Immunoprecipitation with p133 antibody from whole cell extract. (A) Telomerase activity was assayed in supernatants (lanes 1–3) or immunoprecipitates resuspended at 30-fold greater relative concentration (lanes 4–6). Protein A-bound anti-Ct p133 antibody was incubated with extract in the presence of Ct competitor peptide (lanes 2 and 5) or nonspecific peptide AB (lanes 3 and 6), or Protein A-bound random control antibody (R) was incubated with extract in the presence of nonspecific peptide AB (lanes 1 and 4). (B) Telomerase RNA in immunoprecipitates was analyzed by Northern blot. The same volume of bound fractions as in (A) was analyzed (lanes 1–3) in comparison with recombinant telomerase RNA standards (lanes 4–6: 0.1, 0.3, 1.0 ng RNA). (C) Telomerase proteins in immunoprecipitates were analyzed by immunoblot. Three times the volume of bound fraction in (A) was probed for p80 and p95 (lanes 1–3) in comparison with recombinant protein standards (lanes 4–6: 0.25, 1.0, 4.0 ng each protein). The high background in the bound fractions derives from recognition of the primary antibody used for immunoprecipitation.

Figure 3

Immunoprecipitation of p133 from partially purified telomerase fractions. (A–C) Equal relative volumes of supernatants (lanes 1–3) or immunoprecipitates (lanes 4–6) were assayed for telomerase activity (A) or for telomerase RNA by Northern blot (B). Supernatants and an ≈5-fold greater relative amount of immunoprecipitates were analyzed for p133 by immunoblot (C). Protein A-bound polyclonal anti-p80 antibody (lanes 1 and 4), random control antibody (R; lanes 2 and 5), or anti-Ct p133 antibody (lanes 3 and 6) was incubated with extract. The supernatants and immunoprecipitates shown in B were run on separate gels. (D) The immunoprecipitate from a complete activity depletion with anti-Ct p133 antibody was examined by immunoblot for p133, p95, and p80 (lane 1) in comparison with recombinant protein standards (lanes 2–3: 0.25, 1.0 ng each protein, not normalized for relative mass).

Figure 4

Reticulocyte lysate expression of p133 and telomerase RNA. (A) Telomerase activity was assayed for p133 coexpressed with RNA [p133+RNA] (lane 1); individual components expressed alone [p133], [RNA] (lanes 2 and 3); or p133 with motif C substitution of both aspartic acids for alanines coexpressed with RNA [DDp133+RNA] (lane 4). Assays also were performed for lysate-expressed p133 and RNA mixed after synthesis (lane 5); lysate-expressed p133 mixed with purified telomerase RNA (lanes 6–8: 20 ng, 200 ng, 2 μg of RNA); purified RNA alone (lane 9: 2 μg RNA); or partially purified endogenous telomerase (NT, lane 10). The radiolabel in recombinant enzyme lanes in the top half of the gel is from lysate labeling of plasmid DNA. (B) SDS/PAGE followed by autoradiography was used to detect recombinant protein synthesized in the lysates indicated. The migration of molecular mass markers is indicated at right. (C) Northern analysis of telomerase RNA was used to detect recombinant RNA synthesized in 2.5 μl of the lysates indicated. Telomerase RNA standards were loaded at left (1.1, 0.6, 0.3 ng RNA).

Figure 5

Activity of recombinant p133. For each panel, numbers to the left and/or right indicate product sizes in nucleotides. (A and B) The single-stranded DNA primers indicated at top were assayed with coexpressed p133+RNA or partially purified endogenous enzyme (NT). In A, the reaction in lane 4 contained added dATP and dCTP; the reactions in lanes 5 and 9 contained ddTTP instead of dTTP. The exposures of lanes 1–5 and 6–9 were adjusted separately although all lanes were part of the same gel. In B, lane 2 is a marker (M) indicating the migration of an 18- nt telomeric primer extended by addition of one ³²P-dGTP. (C) Primer (G₄T₂)₃ was assayed with coexpressed p133+RNA and increasing concentrations of dGTP (0.6, 2, 6, 20, 60 μM) diluted 6-fold in specific activity from other reactions shown but kept at the same specific activity in the titration. The radiolabel in the top half of the gel is from lysate labeling of plasmid DNA. Note that longer product DNAs are actually less abundant than they appear because they have a higher specific activity. Below each lane, processivity at different dGTP concentrations is compared as the % of 19-nt product (+1) that translocates and is elongated to become 25-nt product (+7), normalized for the 5× greater specific activity of the longer product (i.e., the numbers are a molar percentage of +7 product relative to +1 and +7 products combined). Similar numbers were obtained by maintaining ³²P-dGTP concentration and titrating unlabeled dGTP (data not shown). The stimulatory effect of dGTP appears to saturate between 10 and 100 μM. (D) The full length telomerase RNA (WT) or shorter RNAs (T1–T3) containing the template and different template-adjacent regions as shown were assayed in elongation reactions of the primer (G₄T₂)₃. Purified RNAs (20 pmol) were either added to lysate before synthesis of p133 (lanes 1–4) or added after p133 synthesis (lanes 5–8) subsequent to addition of primer (10 pmol).