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A Human Telomerase Holoenzyme Protein Required for Cajal Body Localization and Telomere Synthesis

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Science  30 Jan 2009:
Vol. 323, Issue 5914, pp. 644-648
DOI: 10.1126/science.1165357

Abstract

Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, including the telomerase reverse transcriptase, the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein 1), that is notably enriched in Cajal bodies, nuclear sites of RNP processing that are important for telomerase function. TCAB1 associates with active telomerase enzyme, established telomerase components, and small Cajal body RNAs that are involved in modifying splicing RNAs. Depletion of TCAB1 by using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association, and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.

The telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC) comprise the minimal catalytic core of the telomerase enzyme (1), whereas dyskerin is an RNA-binding protein that recognizes the H/ACA sequence motif shared by TERC and two groups of noncoding RNAs involved in RNA modification: small Cajal body RNAs (scaRNAs) and small nucleolar RNAs (snoRNAs) (2, 3). Dyskerin functions in part to support telomerase ribonucleoprotein (RNP) biogenesis and TERC stability (4, 5). TERT, TERC, and dyskerin are all components of active telomerase (6), and mutations in any of these genes can cause the human stem cell disorder dyskeratosis congenita (7). Other potential components of active telomerase include three evolutionarily conserved dyskerin-associated proteins, nucleolar protein 10 (NOP10), non-histone protein 2 (NHP2), and glycine/arginine–rich domain containing protein 1 (GAR1) (810), and ever-shorter telomeres 1A (EST1A), a homolog of the yeast telomerase protein Est1p (11, 12). However, the size of active human telomerase, estimated in the 0.65-to-2-MDa range (6, 13, 14), suggests the existence of additional components. We reasoned that other dyskerin-associated proteins may be telomerase components, and we therefore sought to purify dyskerin complexes.

To study dyskerin, we expressed tagged dyskerin protein at endogenous levels in the absence of competing endogenous protein (fig. S1) and isolated dyskerin complexes by using a dual-affinity chromatography strategy. Purified dyskerin complexes were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and nano–liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS) for identification of copurifying proteins (Fig. 1, A and B). Dense peptide coverage was obtained for dyskerin and for the dyskerin-associated adenosine triphosphatases (ATPases) pontin and reptin (14). Each of the evolutionarily conserved dyskerin-binding proteins NHP2, NOP10, and GAR1 was detected, as were the dyskerin-associated proteins nucleolar and coiled-body phosphoprotein of 140 kD. (Nopp140) and nuclear assembly factor 1 (NAF1), a nucleoplasmic factor required for the assembly of H/ACA RNPs, including telomerase (Fig. 1B) (15). In addition, this approach identified WD repeat domain 79 (WDR79) (Fig. 1B), a protein that had not been previously implicated in dyskerin or telomerase function.

Fig. 1.

Identification of TCAB1 as a dyskerin- and telomerase-interacting protein. (A) Dual affinity–purified dyskerin complexes fractionated by SDS-PAGE and silver-stained. (B) Unique and total peptides corresponding to dyskerin-associated proteins identified by nanoLC-MS/MS. (C) Flag-dyskerin interactions with endogenous TCAB1 and telomerase components and (D) Flag-TCAB1 and Flag-NAF1 interactions with endogenous telomerase components. IP–Western blot using extracts from Flag-dyskerin+shRNA cells, Flag-TCAB1+shRNA cells, or Flag-NAF1+shRNA cells is shown. IB, immunoblot; NB, Northern blot; parental, HeLa cells. Plus sign indicates treatment of extracts with RNase A during IP. Recovery control was exogenous RNA spiked into samples after IP to control for differential RNA recovery.

We further characterized WDR79, hereafter referred to as TCAB1 (telomerase Cajal body protein 1) (fig. S2). Endogenous TCAB1 was specifically bound to Flag-dyskerin that was immunoprecipitated from Flag-dyskerin+shRNA HeLa cells (shRNA, short hairpin RNA), as were endogenous pontin, NAF1, TERT, and TERC (Fig. 1C). Interactions between TERT and dyskerin were disrupted by ribonuclease A (RNase A) treatment of the extract, which degraded TERC. In contrast, dyskerin interactions with TCAB1, NAF1, and pontin were not RNase A–sensitive, indicating that these associations occur through protein-protein contacts (Fig. 1C). Reciprocal immunoprecipitation (IP) of Flag-TCAB1 from Flag-TCAB1+shRNA HeLa cells showed that Flag-TCAB1 not only associates with endogenous dyskerin but also with TERT and TERC, the catalytic core of telomerase (Fig. 1D). Like the TERT-dyskerin association, the binding of TERT to Flag-TCAB1 was RNase A–sensitive, suggesting that the interaction of TCAB1 with telomerase is dependent on TERC (Fig. 1D). Thus, TCAB1 interacts specifically with dyskerin, TERT, and TERC, all three known components of active telomerase.

Although TCAB1 associated with TERT, TERC, and dyskerin, it did not interact with the assembly factors NAF1, pontin, or reptin (Fig. 1D), suggesting that TCAB1 may be a component of the enzymatically active telomerase complex rather than a pre-telomerase complex. To test this hypothesis, we asked to what extent telomerase activity in cell extracts was associated with overexpressed TCAB1. Flag-tagged TCAB1, dyskerin, and NAF1 were depleted from extracts by IP (Fig. 2A, lanes 14 to 21). Flag-TCAB1 and Flag-dyskerin immunoprecipitates were associated with high telomerase activity (Fig. 2A, lanes 12 and 13). Moreover, purification of either Flag-TCAB1 or Flag-dyskerin depleted these extracts of telomerase activity, indicating that both Flag-TCAB1 and Flag-dyskerin were associated with nearly all telomerase activity in the extract. In contrast, Flag-NAF1 bound only a small percentage of telomerase and did not deplete telomerase activity from cell extracts (Fig. 2A, lanes 1 to 8). Furthermore, IP of either Flag-TCAB1 or Flag-dyskerin depleted TERC but not unrelated RNAs such as U1 from cell extracts (Fig. 2A, lanes 14 to 21). Flag-TCAB1 and Flag-dyskerin were associated with similar amounts of endogenous TERT and TERC, further suggesting that TCAB1 and dyskerin reside together in the same active telomerase complexes (Fig. 2A, lanes 24 to 25).

Fig. 2.

TCAB1 is a component of active telomerase. (A) Flag-TCAB1 or Flag-dyskerin IP quantitatively codepletes telomerase activity and TERC from extracts. Telomeric repeat amplification protocol (TRAP) assays were performed on extracts before and after IP (lanes 1 to 8) and on each immunoprecipitated complex (lanes 10 to 13). Depletion of tagged proteins and depletion of endogenous TERC on extracts before and after IP are shown in lanes 14 to 21. IP of tagged proteins and associated telomerase components are shown in lanes 22 to 25. U1-splicing RNA was the negative control. (B) IP of endogenous TCAB1 codepletes telomerase activity and TERC. TRAP assays were performed on extracts before and after each IP (lanes 1 to 4) and on the IP (lanes 6 to 8). Depletion of endogenous NAF1 or endogenous TCAB1 and depletion of TERC are shown in lanes 9 to 13. Association of NAF1 and TCAB1 with telomerase components is shown in lanes 14 to 16. Recovery control was exogenous RNA spiked into samples after IP to control for differential RNA recovery.

To understand the composition of endogenous telomerase, IPs were performed using antibodies to TCAB1 and NAF1, each of which efficiently depleted its cognate protein from cell extracts (Fig. 2B, lanes 9 to 13). High telomerase activity was associated with TCAB1 immunoprecipitates, and antibodies to TCAB1 quantitatively depleted telomerase activity from cell extracts (Fig. 2B, lanes 1 to 8, and fig. S4). In contrast, antibodies to NAF1 pulled down only a small percentage of telomerase activity. Assessing the TCAB1 immunoprecipitates for telomerase components revealed that TCAB1 interacts with endogenous dyskerin, TERT, and TERC (Fig. 2B, lanes 14 to 16). IP of TCAB1 effectively depleted cell extracts of TERC by use of Northern blot (Fig. 2B, lanes 9 to 13, and figs. S4 and S5). We conclude that TCAB1, like dyskerin, associates stably with a vast majority of active telomerase and TERC and therefore is a component of a human telomerase holoenzyme.

Using immunofluorescence (IF), we found that stably overexpressed hemagglutinin (HA)–tagged TCAB1 (HA-TCAB1) was distributed weakly throughout the nucleoplasm but was strongly enriched within nuclear foci resembling Cajal bodies, sites of RNP processing shown to contain dyskerin and TERC (1618). IF showed that the HA-TCAB1 foci overlapped with p80-coilin and were therefore Cajal bodies (Fig. 3A). Endogenous TCAB1 was also highly enriched in Cajal bodies, with smaller amounts distributed in the nucleoplasm (Fig. 3B). Although dyskerin accumulates in both Cajal bodies and nucleoli, the Cajal body–restricted localization of TCAB1 suggested that TCAB1 may function specifically with telomerase and with other noncoding RNAs found in Cajal bodies.

Fig. 3.

TCAB1 is enriched in Cajal bodies and associates specifically with scaRNAs. (A) HA-TCAB1 colocalizes with the Cajal body marker p80-coilin. IF using antibodies to HA (rat) and p80-coilin (mouse) on fixed HeLa cells stably overexpressing HA-TCAB1 is shown. (B) Colocalization of endogenous TCAB1 with p80-coilin. IF using antibodies to TCAB1 (rabbit) and p80-coilin (mouse) on fixed HeLa cells. (C) Flag-TCAB1 associates specifically with scaRNAs by IP–Northern blot. Cell extract from parental HeLa cells, Flag-NAF1+shRNA cells, Flag-TCAB1+shRNA cells, or Flag-dyskerin+shRNA cells was incubated with resin coated with antibodies to Flag. RNA was isolated from extracts before (input) and after (depleted) antibody treatment. Two micrograms of total RNA was fractionated by urea-PAGE (lanes 1 to 8), representing 2% of the input. RNAs from IPs are shown in lanes 9 to 12. (D) Endogenous TCAB1 associates specifically with scaRNAs. Antibodies to NAF1 or TCAB1, or the immunoglobulin G control, were used in assays at the same scale as in (C). RNAs from input and depleted extracts are shown in lanes 1 to 5. RNAs from IPs are shown in lanes 6 to 8. Recovery control was exogenous RNA spiked into samples after IP to control for differential RNA recovery.

Immunoprecipitates of Flag-TCAB1 (Fig. 3C) and endogenous TCAB1 (Fig. 3D) were assayed for scaRNAs and representatives of other classes of nuclear noncoding RNAs by Northern blot. Both overexpressed TCAB1 and endogenous TCAB1 specifically bound H/ACA scaRNAs but associated with neither snoRNAs nor splicing RNAs. In contrast, Flag-dyskerin specifically bound both H/ACA scaRNAs and H/ACA snoRNAs, whereas NAF1 did not associate stably with any noncoding RNAs studied. For each H/ACA scaRNA tested, a substantial proportion was depleted from the extract by IP of TCAB1 (Fig. 3, C, lanes 5 to 6, and D, lanes 1 to 5). Thus, TCAB1 specifically binds scaRNAs, which share a Cajal body (CAB) box sequence that controls Cajal body localization, and provides a potential mechanism to explain how scaRNAs, including TERC, are retained in Cajal bodies.

TCAB1 depletion by using retroviral-encoded shRNAs reduced neither telomerase activity nor TERC levels, in contrast to dyskerin depletion, which markedly diminished TERC and telomerase activity (Fig. 4A). Thus, TCAB1 may be required in vivo for a step after the assembly of a catalytically competent telomerase complex. To determine the role of TCAB1 in Cajal body localization of telomerase, TERC localization was measured by RNA fluorescence in situ hybridization (FISH) in TCAB1-depleted HeLa cells. FISH revealed that TCAB1 knockdown substantially reduced the percentage of cells in which TERC was found in Cajal bodies (Fig. 4, B and C) without affecting overall TERC RNA levels (Fig. 4A). Cajal bodies have been directly implicated in the delivery of TERC to telomeres during S phase (1921). Therefore, we also examined the effect of TCAB1 knockdown on the localization of TERC to telomeres, using FISH for TERC and IF for telomere repeat binding factor 2 (TRF2). Depletion of TCAB1 substantially reduced the presence of TERC at telomeres during S phase (Fig. 4, B and C). Thus, TCAB1 is required for TERC localization in Cajal bodies and for delivery of TERC to telomeres during S phase.

Fig. 4.

TCAB1 is essential for TERC localization to Cajal bodies and for telomere synthesis by telomerase. (A) HeLa cells were transduced with retroviruses expressing independent shRNA sequences targeting the indicated proteins or with empty vector control. Telomerase activity was measured by TRAP assay. (B) TERC colocalization with p80-coilin was determined by RNA FISH for TERC (green) and IF for p80-coilin (red) (top). Cells synchronized in S phase were assayed for TERC by RNA FISH (green) and for telomeres with antibody to TRF2 (red) to assess trafficking of TERC to telomeres (bottom). (C) Quantification of data in (B). (Top) Cells in which TERC colocalized with p80-coilin (+) versus cells in which TERC was not detected in Cajal bodies (–) (P < 0.0001, Fisher's exact test). (Bottom) Cells in which TERC colocalized with telomeres (+) versus cells in which TERC was not detected at telomeres (–)(P < 0.0001, Fisher's exact test). (D) Telomere lengths were measured using terminal restriction fragment (TRF) Southern blot in HTC75 cells overexpressing wild-type TERC (lanes 1 to 8) or mutant TERC-m1 (lanes 9 to 12). Cells overexpressing wild-type TERC were transduced with shRNA retroviruses targeting TCAB1 or with the empty vector. (E) Effect of TCAB1 depletion on endogenous telomerase was assessed by TRF Southern blot in parental HTC75 cells that were transduced with the empty vector or retroviruses expressing independent TCAB1 shRNAs. Cells were counted at each passage and population doublings are indicated. (F) Model for TCAB1 function in the telomere synthesis pathway.

To determine whether TCAB1 is required for telomere addition by telomerase, we first induced telomere elongation through TERC overexpression in HTC75 fibrosarcoma cells. Overexpression of wild-type TERC lengthened telomeres with cell passage, but telomere lengthening was inhibited in cells overexpressing a CAB box–mutant TERC that fails to accumulate in Cajal bodies, as previously shown (19). Depletion of TCAB1 in cells overexpressing wild-type TERC substantially inhibited telomere elongation, mimicking the effect of the CAB box mutation (Fig. 4D and fig. S6). To determine whether TCAB1 is required for telomere synthesis by endogenous telomerase, we assayed telomere lengths with serial passage in HTC75 cells transduced with TCAB1 shRNAs or with the empty vector. Both shRNAs targeting TCAB1 led to progressive telomere shortening as compared with that of the empty vector control (Fig. 4E and fig. S7), indicating that TCAB1 is required for telomere synthesis in human cancer cells.

Our data identify TCAB1 as a Cajal body–enriched protein that associates with TERC and other scaRNAs and explains how TERC, and perhaps other scaRNAs, localize in Cajal bodies. TCAB1 functions as a telomerase holoenzyme component in the telomere synthesis pathway at a step after the assembly of a minimal telomerase complex containing TERT, TERC, and dyskerin. NAF1 and the ATPases pontin and reptin are required for assembly of the catalytically competent complex (14, 15). In contrast, TCAB1 stably associates with active telomerase enzyme and directs it through Cajal bodies to telomeres. In this manner, TCAB1 may act as a Cajal body–targeting or –retention factor, may facilitate additional assembly steps of the enzyme in Cajal bodies, and/or may facilitate translocation of telomerase to telomeres (Fig. 4F). The interaction of telomerase with telomeres and the activity of the telomerase holoenzyme may be enhanced by the telomere-binding proteins TPP1 and protection of telomeres 1 (POT1) (22, 23), as well as other factors that remain to be discovered.

Supporting Online Material

www.sciencemag.org/cgi/content/full/323/5914/644/DC1

Materials and Methods

Figs. S1 to S7

References

References and Notes

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