Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly

Abstract

Telomerase is a multisubunit ribonucleoprotein (RNP) complex that adds telomere repeats to the ends of chromosomes. Three essential telomerase components have been identified thus far: the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC), and the TERC-binding protein dyskerin. Few other proteins are known to be required for human telomerase function, limiting our understanding of both telomerase regulation and mechanisms of telomerase action. Here, we identify the ATPases pontin and reptin as telomerase components through affinity purification of TERT from human cells. Pontin interacts directly with both TERT and dyskerin, and the amount of TERT bound to pontin and reptin peaks in S phase, evidence for cell-cycle-dependent regulation of TERT. Depletion of pontin and reptin markedly impairs telomerase RNP accumulation, indicating an essential role in telomerase assembly. These findings reveal an unanticipated requirement for additional enzymes in telomerase biogenesis and suggest alternative approaches for inhibiting telomerase in cancer.

Figure 1. Pontin and reptin co-purify with TERT through dual affinity purification and are components of a large TERT complex

(A) Sedimentation of endogenous telomerase and Flag-TERT complexes. Extracts from HeLa cells (left) and HeLa-Flag-TERT cells (right) were fractionated through 10-30% glycerol gradients. Total protein across the gradient was measured by Bradford assay (top). IB, immunoblot; NB, northern blot. (B) Coomassie stain of affinity-purified TERT complexes fractionated by SDS-PAGE. (C) Diagram of pontin and reptin proteins shows location of unique peptides identified by MS (black bars). Blue boxes denote ATPase domains. (D) Number of unique peptides obtained by mass spectrometry from four independent TERT purifications. (E) Immunoprecipitation of Flag-TERT complexes after sedimentation in 1A. Adjacent fractions were pooled and immunoprecipitated. Pontin and reptin association with TERT peaked in fractions 13-16.

Figure 2. Pontin and reptin interact with endogenous TERT and TERC

(A) Western blot showing genetic co-depletion of endogenous pontin and reptin with shRNA specifically targeting either protein. Control shRNA had no effect on pontin or reptin levels. Western for Brg-1 was used as a loading control. Independent shRNAs are denoted: A,B,C. (B) Flag-pontin expressed by retroviral transduction under-accumulates relative to endogenous levels (lane 5). Serial transduction with shRNA-resistant Flag-pontin followed by shRNA against endogenous pontin, results in depletion of endogenous pontin and accumulation of Flag-pontin to endogenous levels by pontin western blot (compare lanes 1, 3). Cells treated in this manner are referred to as Flag-pontin^+shRNA cells. A similar strategy was used to generate Flag-reptin^+shRNA cells. (C) Endogenous TERT is detected in pontin and reptin complexes purified from Flag-pontin^+shRNA and Flag-reptin^+shRNA cells. Flag immunoprecipitation was followed by western blot with anti-TERT antibodies. RNase A treatment during immunoprecipitation did not reduce TERT association. (D) Pontin and reptin interact with endogenous TERT and with endogenous TERC in a largely TERT-dependent manner. Flag-pontin^+shRNA and Flag-reptin^+shRNA cells treated with independent shRNA vectors targeting TERT (A and B) resulted in decreased association of pontin and reptin with TERT and TERC. A recovery control RNA was spiked into each IP-northern blot sample to control for differential nucleic acid extraction.

Figure 3. Pontin and reptin are required for telomerase activity and for TERC accumulation

(A) Diminished telomerase activity by pontin shRNA in HeLa cells. (B) Titration of protein extracts from (A). Telomerase activity is reduced to 10-20% of wild-type levels by pontin shRNA B or C sequences. (C) TERC depends on pontin and reptin for accumulation to wild-type levels. Pontin shRNA reduced TERC to approximately 25% of wild-type levels. Band intensities for TERC and U3 snoRNA were quantified and presented as a fraction of the loading control U1 snRNA. (D) ATPase activity of pontin is required for maintenance of TERC levels. HeLa cells depleted of pontin using shRNA are rescued by expression of a wild-type Flag-pontin construct, but not the ATPase-mutant Flag-pontin^D302N. Note that both pontin cDNAs contained silent mutations rendering them insensitive to the pontin A shRNA. Band intensities were quantified as in 5C. (E) Endogenous TERT-TERC association is compromised following pontin and reptin depletion. Immunoprecipitation using anti-TERT antibodies in HeLa cells treated with shRNA to pontin reveal decreased TERT-TERC association relative to control shRNA vectors. (F) Experiment in 3E was repeated in HeLa-Flag-TERT cells using anti-Flag resin to immunoprecipitate Flag-TERT.

Figure 4. Pontin and reptin interact with dyskerin and are required for dyskerin accumulation

(A) HA-dyskerin associates with either Flag-pontin or Flag-reptin by anti-Flag immunoprecipitation from co-transfected cells. Flag-BAF57 was used as a negative control. (B) Flag-dyskerin co-immunoprecipitates endogenous pontin and reptin in transfected 293T cells. HA-TERT interacts weakly with Flag-dyskerin, but shows enhanced binding by co-expression of TERC. Flag-p53 serves as a negative control. The white asterisk indicates mouse IgG heavy chain. (C) Dyskerin, pontin, and reptin interact at the endogenous level in an RNase A-insensitive manner. Anti-Flag immunoprecipitation in Flag-pontin^+shRNA and Flag-reptin^+shRNA cells readily recovers endogenous dyskerin and TERT. Extracts from HeLa cells stably expressing Flag-TERT were immunoprecipitated with anti-Flag resin in parallel. RNase A treatment significantly reduced the amount of dyskerin associated with Flag-TERT, but did not alter the amount of dyskerin bound to pontin or reptin. Asterisk indicates a non-specific band detected by western of whole cell lysate with anti-TERT antibodies. (D) Suppression of pontin or reptin with shRNA impairs dyskerin accumulation (right panels), but shRNA vectors targeting dyskerin affect steady state levels of neither pontin nor reptin (left panels).

Figure 5. Pontin interacts directly with dyskerin and TERT

(A) Illustration of TERT fragments used for binding assays. Results from 5B are scored in the right column as “P/R binding.” (B) Transfection assays in 293T show that Flag-tagged fragments of TERT incorporating the central reverse transcriptase domains efficiently co-immunoprecipitate endogenous pontin and reptin. Flag-p53 was used as a negative control. (C) Rabbit reticulocyte lysate transcription-translation of Flag-tagged dyskerin, TERT, and TERT fragments co-immunoprecipitate rabbit pontin intrinsic to the lysate. (D) Coomassie stained gels of bacterially expressed and purified Flag-pontin, MBP-TERT fragments, and MBP-dyskerin purified for direct binding assay in 5E. (E) Direct binding assay between Flag-pontin and MBP-tagged proteins. MBP-tagged proteins were immobilized with amylose resin and incubated with Flag-pontin, washed, and analyzed by western blot with anti-Flag antibodies. Ponceau S stain shows relative amount of MBP-tagged proteins. (F) Illustration of pontin fragments used for binding assays. Ribbon diagram for a pontin monomer (PDB 2C9O; (Matias et al., 2006)) guided the design for our pontin fragments. Results from 5G are scored in the right column as “dyskerin binding”. (G) Co-transfection of HA-dyskerin with Flag-pontin fragments in 293T cells show that the C-terminal fragment of pontin (pontin-C) is sufficient to co-immunoprecipitate HA-dyskerin. Flag-p53 was used as a negative control.

Figure 6. Pontin and reptin interact with TERT in S phase

(A) Flow cytometry analysis of DNA content in HeLa-Flag-reptin^+shRNA cells released from double thymidine blockade over a ten hour time course. Cells were released from the second block and harvested at two hour intervals, and a portion of cells were fixed and stained with propidium iodide to monitor synchrony. The zero hour time point corresponds to unreleased cells. (B) Co-immunoprecipitation of reptin with TERT and dyskerin over a ten hour timecourse. The association of reptin with pontin and dyskerin is constant across the cell cycle, whereas associated TERT peaks in S phase. Band intensities were quantified and displayed as fold change in the accompanying graph. Western blot for PCNA was used as an independent marker of S phase. (C) Flow cytometry analysis of DNA content in HeLa-Flag-reptin^+shRNA cells synchronized using double thymidine blockade as in 6A, except cells were harvested at four hour intervals over a 24 hour time course. (D) Co-immunoprecipitation of reptin with pontin, dyskerin, and TERT though a 24 hour timecourse following synchronization shown in 6C. Band intensities were analyzed as in 6B. Note that the amount of TERT in the reptin complex increases during the second S phase with kinetics similar to PCNA.

Figure 7. TERT exists in multiple telomerase complexes

(A) Western blot analysis of whole cell lysates prepared from “replacement” cell lines for pontin, reptin, and dyskerin. Retrovirally introduced Flag-tagged pontin, reptin, or dyskerin accumulates to endogenous levels upon depletion of the respective endogenous proteins with shRNA. Note that Flag-pontin, -reptin, and -dyskerin coding sequences contain silent mutations rendering them insensitive to the shRNA vectors. (B) Whole cell lysates in 7A were depleted of the Flag-tagged protein using anti-Flag resin. Depletion was assessed by western blot with anti-Flag antibodies. (C) TRAP assay of extracts in 7B shows that immunoprecipitation of Flag-TERT and Flag-dyskerin, but not Flag-pontin or Flag-reptin, depletes telomerase activity. (D) TRAP assay on immunoprecipitates from 7C. Reptin co-immunoprecipates a small but reproducible amount of telomerase activity. (E) TERT associates similarly with pontin/reptin and dyskerin by immunoprecipitation-western blot. Isolated pontin and reptin complexes have low TRAP activity but substantial TERT protein (shown in 7D). (F) Model for telomerase assembly facilitated by pontin and reptin. Pontin and reptin are required for accumulation of a TERC- and dyskerin-containing RNP through steps which require ATPase function (bottom). Pontin and reptin also bind TERT (top) and may help to bring together or remodel a nascent TERT/TERC/dyskerin complex through a stepwise process. Other factors and/or ATP hydrolysis may convert the complex into the enzymatically active, TRAP-positive telomerase complex. At this point, pontin and reptin may dissociate from telomerase or remain associated with a telomerase complex that has low activity in vitro. See discussion for further details.