The Euplotes telomerase subunit p43 stimulates enzymatic activity and processivity in vitro

Abstract

Telomerase is a reverse transcriptase that synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. Although it is minimally composed of a conserved catalytic protein subunit (TERT) and an RNA component, additional accessory factors present in the holoenzyme play crucial roles in the biogenesis and function of the enzyme complex. Telomerase from the ciliate Tetrahymena can be reconstituted in active form in vitro. Using this system, we show that p43, a telomerase-specific La-motif protein from the ciliate Euplotes, stimulates activity and increases repeat addition processivity of telomerase. Activity enhancement by p43 requires its incorporation into a TERT.RNA.p43 ternary complex but is independent of other dissociable protein factors functioning in telomerase complex assembly. Stimulation is enhanced at elevated temperatures, supporting a role for p43 in structural stabilization of a critical region of the RNA subunit. To our knowledge, this represents the first demonstration that an authentic telomerase accessory protein can directly affect the enzymatic activity of the core enzyme in vitro.

FIGURE 1.

Secondary structures of telomerase RNAs from (A) Euplotes aediculatus (Lingner et al. 1994) and (B) Tetrahymena thermophila (Romero and Blackburn 1991; ten Dam et al. 1991; McCormick-Graham and Romero 1995). Experimentally determined sites of binding of p43 (Aigner et al. 2003) and TERT (Licht and Collins 1999; Lai et al. 2003) are outlined with solid ovals, and the inferred binding site of p43 in B is depicted by an oval outlined by a dashed line.

FIGURE 2.

A model for Tetrahymena telomerase action. Binding of a telomeric oligonucleotide, for instance, 5′-(G₄T₂)₃-3′, to the enzyme involves base pairing of the 3′ end of the primer with the template region of telomerase RNA (A). Upon addition of substrate dNTPs, telom-erase catalyzes the RNA-templated extension of the primer to the 5′ end of the template region (B). At this point, the extended primer may translocate to the alignment region at the beginning of the template (C) to facilitate a new round of nucleotide addition (D), thus processively extending the primer until it dissociates from the enzyme. Product dissociation (indicated by open arrows) predominantly occurs when synthesis has reached the end of the template, either prior (E,F) or subsequent (G) to translocation. This mechanism of telomerase action results in a characteristic ladder of extension products that differ in size by 6 nt.

FIGURE 3.

Euplotes p43 forms a stable ternary complex with TERT and telomerase RNA from Tetrahymena. (A) FLAG-tagged TERT (lanes 2,3) or untagged TERT (lanes 4–7) and FLAG-tagged p43 (lanes 5,6) or untagged p43 (lanes 2–4, 7) were separately translated in RRLs in the presence or absence of ³²P-labeled telomerase RNA, as indicated. TERT- and p43-containing RRLs were mixed, incubated to allow binding, and subjected to anti-FLAG immunoprecipitation. Bound material was released from the beads, separated on an SDS gel, and the RNA visualized with a PhosphorImager. (Lane 1) 10% of the RNA used for immunoprecipitation. (B) RRLs containing ³⁵S-labeled TERT and FLAG-tagged p43 were combined and unlabeled telomerase RNA was added at the relative concentrations given above the gel, whereas the concentrations of full-length p43 and TERT were held constant at ~12 nM each. The Input column shows the indicated fractions of the material used for immunoprecipitation. p43 is actually a 51-kDa protein (Aigner et al. 2000). Full-length Tetrahymena TERT is a 133-kDa protein but in vitro translation produces significant amounts of shorter fragments that do not bind RNA. (M) ¹⁴C-labeled protein markers of the indicated molecular masses (in kDa).

FIGURE 4.

p43 enhances both overall telomerase activity and repeat addition processivity. Purified recombinant p43 expressed in insect cells (lanes +) or p43 buffer as a control (lanes -) was incubated with telomerase RNA, followed by addition of RRL containing ³⁵S-labeled T7-tagged TERT. Relative levels of (A) telomerase activity and (B) TERT protein in the samples were determined prior to (left panels) or after (right panels) immunopurification on T7-antibody beads. In A, the number of telomeric repeats added to the primer are indicated on the left, and the total number of nucleotides added is indicated on the right. (LC) Loading control; a radiolabeled 100-nt DNA added prior to the telomerase assay sample work-up to account for variability in recovery and gel loading. Quantitation of telomerase activity and processivity are summarized below the gel in A. For determination of the overall nucleotide incorporation activity of telomerase (“Activity”), total radioactivity in each telomerase assay lane (panel A) was quantitated, corrected for TERT levels (panel B) and for gel loading, and normalized to the activity seen in the second lane. Repeat addition processivity (“Processivity”) was quantitated as follows. The intensities of the major repeat bands (indicated in A) were normalized to the intensity of the first repeat, adjusted for specific activity, and plotted against the repeat number. Fitting of the data to a curve of the form y = C × e^−x yields a log-linear relationship, and processivity is expressed as the inverse of the exponent x of the curve fit (a measure of the slope of the curve), normalized to the value of x⁻¹ from the second lane. (C) Graphic representation of telomerase processivity. Data were plotted as described for A. (Solid and open diamonds) Data from samples in the presence and absence of p43, respectively, before immunoprecipitation. (Solid and open squares) Data from samples in the presence and absence of p43, respectively, after immunoprecipitation.

FIGURE 5.

The extent of telomerase stimulation by p43 depends on the p43 concentration and the order of ternary complex assembly. (A) RRLs containing p43 (lanes +) or control RRL (lanes -) were mixed with RRLs containing preformed complexes consisting of telomerase RNA and RRL-expressed TERT at the indicated molar ratios of p43:TERT and assayed for telomerase activity. The concentration of TERT decreased at high ratios of p43:TERT, resulting in decreased overall activity. (B) RRLs containing TERT and RRLs containing preformed complexes consisting of telomerase RNA and RRL-expressed p43 (lanes +) or telomerase RNA only (lanes -) were mixed at the indicated molar ratios of p43:TERT and assayed for telomerase activity. At low p43:TERT, RNA is limiting and at very high p43:TERT, TERT is limiting, resulting in decreased activity at the high and low ends. (C) Quantitation of two such p43 titrations. For each titration point, the level of telomerase activity in the presence of p43 relative to that in the absence of p43 was determined and plotted against the molar ratio of p43:TERT. (Open diamonds) p43 added to preformed complexes of TERT and telomerase RNA (as described in A); (solid diamonds) TERT added to preformed complexes of p43 and telomerase RNA (as described in B). (Dashed and solid lines) Log-linear fits of the datapoints denoted by the open and solid symbols, respectively.

FIGURE 6.

Binding of p43 does not protect telomerase RNA against degradation during the assay. Trace-³²P-labeled telomerase RNA at the concentration used in a standard telomerase reaction was incubated in RRLs expressing no protein (A), p43 (B), TERT (C), or in RRL coexpressing p43 and TERT (D). After translation for 1 h, the RRLs were incubated at 30°C and on ice for 30 min each to allow complex formation, followed by a mock assay for telomerase activity for 1 h at 30°C. Aliquots were removed at 30-min intervals, subjected to gel electrophoresis, and analyzed using a PhosphorImager. (E) Quantitation of the data in A–D. The amount of RNA present at each timepoint was normalized to the amount of RNA at time = 0 min and plotted against the time of incubation.

FIGURE 7.

The stimulatory effect of p43 on telomerase activity is most pronounced at slightly elevated temperature. (A) Telomerase complexes were assembled with and without p43 as described in the legend to Figure 4 ▶ and assayed at the indicated temperatures. (B) Quantitation of the data shown in A. Mean telomerase activities (± standard deviations) in the presence (gray columns) and absence (white columns) of p43 were determined at the indicated temperatures in three independent experiments and are expressed relative to the mean telomerase activity measured at 30°C in the absence of p43. (Asterisks) Statistically significant (p < 0.01, Student’s t test) differences in activity. The line represents the ratios of telomerase activities with p43 relative to those without p43 at the indicated temperatures.