Telomerase trafficking and assembly in Xenopus oocytes

Abstract

The core components of telomerase are telomerase RNA (TR) and telomerase reverse transcriptase (TERT). In vertebrate cells, TR and TERT have been reported to associate with intranuclear structures, including Cajal bodies and nucleoli as well as telomeres. Here, we examined the time course of both TR localization and assembly of TR with TERT in Xenopus oocytes. The major trafficking pathway for microinjected TR is through Cajal bodies into the nucleoplasm, with a fraction of TR found in nucleoli at later time points. Telomerase assembly precedes nucleolar localization of TR, and TR mutants that do not localize to nucleoli form active enzyme, indicating that localization of TR to nucleoli is not required for assembly with TERT. Assembly of telomerase coincides with Cajal-body localization; however, assembly is also unaffected by a CAB-box mutation (which significantly reduces association with Cajal bodies), suggesting that Cajal-body localization is not important for assembly. Our results suggest that assembly of TR with TERT occurs in the nucleoplasm. Unexpectedly, however, our experiments reveal that disruption of the CAB box does not eliminate early targeting to Cajal bodies, indicating that a role for Cajal bodies in telomerase assembly cannot be excluded on the basis of existing knowledge.

Fig. 1.

Injected xTR assembles into active telomerase enzyme in Xenopus oocytes. (A) The secondary structure of xTR. Structure modified from Chen et al. (Chen et al., 2000). Substitution mutations were made in the template region, and the box H and box ACA of the box-H/ACA motif. (B) Injected xTR associates with endogenous xTERT to form active enzyme. Altered-template xTR was injected into oocytes and a modified TRAP assay specific to this injected xTR was performed. Internal control for PCR reaction is indicated (IC). Activity (ladder of bands above IC) is observed upon injection of this xTR (Alt. template) and is lost with RNAse A treatment (RNAse). Using the modified TRAP assay, activity was not detected in uninjected cells (uninjected) or in cells injected with xTR containing a wild-type template (WT). (C) Assembly occurs in vivo, not in vitro. Altered-template xTR was injected into Xenopus oocytes and the nuclei were dissected 1 hour after injection and transferred to lysis buffer (in vivo). The same amount of RNA was added into lysis buffer and incubated on ice for 30 minutes with uninjected nuclei (in vitro). The modified TRAP assay was performed on these lysates and uninjected control lysate (uninjected).

Fig. 2.

Injected xTR rapidly associates with nuclear structures before accumulating in the nucleoplasm in Xenopus oocytes. (A) Framework of the experiments in this manuscript. xTR (wild-type or a sequence variant) is injected into Xenopus oocyte nuclei. At specific time points following injection, nuclei are isolated and analyzed. Three tests – biochemical fractionation, nuclear spreads and TRAP assays – were performed to detect the distribution (in subnuclear structures or nucleoplasm), subnuclear localization and activity of the injected RNA, respectively. (B) Distribution of injected radiolabeled xTR in Xenopus oocytes. ³²P-labeled xTR and control RNAs U3 and U1 were co-injected into oocyte nuclei. The nuclei were obtained and biochemical fractionation was performed at the indicated time points. The RNAs present in structural (pellet) and nucleoplasmic (supernatant) fractions are shown. U3 is a snoRNA that stably associates with nucleoli. U1 is a snRNA found primarily in the nucleoplasm. (C) Graph of the distribution of xTR with relation to U3 and U1 control RNAs. The results in B were quantitated and the percent (%) of each indicated RNA found in the pellet is graphed relative to time (in hours) after injection. The results of three independent experiments are plotted; error bars indicate the standard deviation.

Fig. 3.

xTR rapidly localizes to Cajal bodies following injection into Xenopus oocytes. Fluorescein-labeled xTR (xTR panels) was injected (1 fmol/cell) and nuclear spreads were prepared at the indicated time points (hours) after injection. Cajal bodies were immunostained with anti-coilin antibodies (coilin panels) and are indicated with arrowheads. Uninj, uninjected nucleus; DIC, differential interference contrast. The larger structures present in the DIC field are nucleoli. Scale bar: 10 μm.

Fig. 4.

Nucleolar localization of xTR is not required for enzyme assembly. (A) Box-H- and box-ACA-mutant xTRs still assemble into an active enzyme in vivo. Oocyte nuclei were harvested 1 and 3 hours after injection of ³²P-labeled altered-template wild-type (WT) and mutant (H and ACA) xTRs. The nuclear extract was then subjected to the modified TRAP assay. IC, internal control for TRAP assay; –, no lysate control; uninj, uninjected nuclei. (B) Box-H- and box-ACA-mutant xTRs are less stable than wild-type xTR. The RNAs present in the oocyte nuclei from A were isolated and analyzed by gel electrophoresis. U3 served as an injection and loading control. M, marker for amount of RNA injected. (C) Box-ACA-mutant xTR is assembled into active telomerase complexes more slowly than wild-type xTR. Box-ACA-mutant xTR (1 fmol/cell) and a range of levels of wild-type xTR (1, 0.2, 0.04 and 0.01 fmol/cell) were injected into Xenopus nuclei (all RNAs had altered template regions). The nuclei were isolated at 1 and 3 hours after injection and modified TRAP assays were performed on the nuclear lysates. At each time, RNAs were extracted and analyzed by gel electrophoresis (bottom panel). Arrows denote levels of wild-type and box-ACA-mutant RNA that produce comparable activity at 1 and 3 hours. (D) Box-H/ACA-mutant xTRs do not localize to nucleoli, but do associate with Cajal bodies. Fluorescently labeled wild-type (WT panels) or mutant (H and ACA panels) xTR was injected into Xenopus oocytes (2 fmol/cell) and nuclear spreads were prepared 1 hour after injection (xTR panels). Cajal bodies were immunostained with anti-coilin antibodies (coilin panels) and are indicated with arrowheads. DIC, differential interference contrast. The large spherical structures seen in the DIC panels correspond to nucleoli. Scale bar: 10 μm.

Fig. 5.

Telomerase assembly is rapid and the active enzyme is distributed mainly in the nucleoplasm. (A) Injected xTR assembles rapidly with endogenous TERT. Altered-template xTR was injected into oocyte nuclei (1 fmol/cell). At the indicated times (in hours) after injection, nuclei were collected and fractionated. The resulting supernatants and pellets were subjected to modified TRAP assay (specific for the injected xTR). Unfractionated nuclear extracts (total) were analyzed in tandem. −, negative control (no lysate); uninj, uninjected oocytes; IC, internal control for TRAP assay. (B) Endogenous telomerase activity resides in the nucleoplasm. Uninjected nuclei were fractionated, and telomerase activity in the pellet (P) and supernatant (S) were detected using the standard TRAP assay. Activity in total (not fractionated) extract (T) was analyzed as well. The distribution of xTR, U3 and U1 RNAs from the same fractionation was visualized by northern analysis in the bottom panel.

Fig. 6.

CAB-box-mutant xTR assembles with endogenous TERT and associates transiently with Cajal bodies. (A) The CAB-box mutant is retained within the nucleus and assembles with TERT. A total of 1 fmol of wild-type (WT) or CAB-motif-mutant (CAB) xTR was injected into the nuclei of Xenopus oocytes. (Both RNAs contained the mutant template region.) The activity (detected by modified TRAP assay, top panel), stability and nucleocytoplasmic distribution (bottom panel) of each RNA was analyzed 1 hour after injection. U3 RNA serves as an injection and loading control. Uninj, uninjected oocyte; IC, internal control; M, marker for amount of RNA injected; N, nuclear; C, cytoplasmic. (B) The subnuclear distribution of CAB-box-mutant xTR varies from that of wild type. Wild-type (WT) or CAB-box-mutant (CAB) xTR was co-injected with U3 and U1 control RNAs. Biochemical fractionation experiments were performed as described in Fig. 2. The percentage of each RNA present in the pelleted fraction (corresponding to subnuclear structures) is plotted versus time following injection (in hours). The graph represents the results of two independent experiments; error bars indicate standard deviation. (C) CAB-box-mutant xTR initially localizes to Cajal bodies prior to associating with nucleoli. A total of 1 fmol of fluorescently labeled wild-type or CAB-box-mutant xTR was injected (xTR panels). Nuclear spreads were prepared 1 and 6 hours after injection (as indicated). Cajal bodies were immunostained with anti-coilin antibodies (coilin panels, denoted by arrowheads). Uninj, uninjected nuclei; DIC, differential interference contrast. The larger spherical structures present in the DIC panel are nucleoli. Scale bar: 10 μm.