Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells - PubMed (original) (raw)

Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells

Rebecca L Tomlinson et al. Mol Biol Cell. 2008 Sep.

Abstract

Telomere maintenance by telomerase is critical for the unlimited division potential of most human cancer cells. The two essential components of human telomerase, telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT), are recruited from distinct subnuclear sites to telomeres during S phase. Throughout the remainder of the cell cycle hTR is found primarily in Cajal bodies. The localization of hTR to Cajal bodies and telomeres is specific to cancer cells where telomerase is active and is not observed in primary cells. Here we show that the trafficking of hTR to both telomeres and Cajal bodies depends on hTERT. RNA interference-mediated depletion of hTERT in cancer cells leads to loss of hTR from both Cajal bodies and telomeres without affecting hTR levels. In addition, expression of hTERT in telomerase-negative cells (including primary and ALT cancer cell lines) induces hTR to localize to both sites. Factors that did not stimulate hTR localization in our experiments include increased hTR RNA levels and Cajal body numbers, and expression of SV40 large T antigen and oncogenic Ras. Our findings suggest that the trafficking of telomerase to Cajal bodies and telomeres in cancer cells correlates with and depends on the assembly of the enzyme.

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Figures

Figure 1.

Localization of hTR to intranuclear foci is not stimulated by increased levels of hTR or Cajal bodies. (A) Ectopic overexpression of hTR does not lead to localization of hTR to nuclear foci. IMR90 primary fibroblasts transiently transfected with an hTR construct (Li et al., 2004), VA13, and VA13 cells stably expressing the same construct were analyzed by hTR FISH. GFP marks cells transfected with the hTR construct. Nuclei are stained with DAPI. (B) Comparison of hTR levels. RNase protection assay on total RNA from HeLa, IIICF-T/C3, GM847, VA13, and VA13+hTR cells. Note that hTR sometimes migrates as a doublet. U3 snoRNA and U1 snRNA were used as loading controls. (C) Increasing Cajal body numbers does not promote hTR localization. MCF7 breast cancer cells and human mammary epithelial (HME) primary cells were grown at 37°C (normal growth temperature) or 32°C (to increase Cajal body formation). Cells were costained by hTR FISH (hTR) and coilin IF (marker protein for Cajal bodies, coilin). Filled arrowheads, Cajal bodies that contain hTR; open arrowheads, Cajal bodies without hTR. (D) hTR does not localize to Cajal bodies in the ALT cell lines GM847 and IIICF-T/C3. HeLa, GM847, and IIICF-T/C3 cells were coanalyzed for hTR (detected by FISH, hTR) and coilin localization (detected by IF, coilin). Filled arrowheads denote Cajal bodies that contain hTR; open arrowheads indicate Cajal bodies without hTR. DIC, differential interference contrast. Scale bars, 10 μm.

Figure 2.

hTERT expression leads to accumulation of hTR within nuclear foci. (A) MCF7 breast cancer cells, human mammary epithelial (HME) normal cells, and HME cells expressing combinations of hTERT, large T antigen, and oncogenic (onc.) Ras were subjected to hTR FISH (hTR). Nuclei are stained with DAPI. Arrowheads denote hTR foci. Scale bar, 10 μm. (B) RNase protection assay on total RNA from HME and HME+hTERT cells. U3 snoRNA and U1 snRNA were used as loading controls.

Figure 3.

Expression of hTERT induces hTR localization to Cajal bodies. HME and IMR90 cells that ectopically express hTERT were cultured at 32°C to increase Cajal body numbers. These cells and GM847 (ALT) cells that stably express hTERT were analyzed by hTR FISH (hTR) and coilin IF (coilin). Arrowheads indicate hTR foci that colocalize with coilin (Cajal bodies). Scale bars, 10 μm.

Figure 4.

hTERT expression is necessary for localization of hTR to Cajal bodies. (A and B) RNA interference mediated depletion of hTERT leads to a loss of hTR within Cajal bodies. HeLa cells were transfected with hTERT shRNA (middle and bottom rows) or an empty vector control (top row). (A) Cells were coanalyzed for hTERT and coilin localization (detected by IF); (B) hTR (detected by FISH) and coilin localization (detected by IF). Data in each set of panels were normalized relative to the empty vector control to allow direct visual comparison. Filled arrowheads, hTR in Cajal bodies; open arrowhead, a Cajal body with reduced level of hTR. DAPI was used to stain the DNA. Scale bar, 10 μm. (C) RNA interference mediated depletion of hTERT does not affect hTR levels. RNase protection assay on total RNA from HeLa cells transfected with hTERT shRNA or empty vector. Note that hTR sometimes migrates as a doublet. U3 snoRNA and U1 snRNA are used as loading controls.

Figure 5.

hTR foci in normal cells that express hTERT occur primarily in S phase. (A) hTR foci are predominantly found in S phase cells. IMR90 and IMR90+hTERT cells were subjected to hTR FISH (hTR) and BrdU labeling (BRDU). Arrowheads indicate hTR foci, which are present in BrdU-positive IMR90+TERT cells. Note that the BrdU-negative IMR90+hTERT cell in the middle row of panels does not contain hTR foci. DIC, differential interference contrast. Scale bar, 10 μm. (B) The percentage of cells with hTR foci increases in S phase the percentage of cells with hTR foci. The percentage of IMR90+hTERT cells with hTR foci is graphed relative to time after release from a double thymidine block. Data are compiled from over 100 cells and two independent experiments. A, asynchronous cells.

Figure 6.

hTERT expression is sufficient to stimulate localization of hTR to telomeres. (A) hTR localizes to a subset of telomeres in normal cells that ectopically express hTERT. IMR90+ hTERT (top panels) and HME+hTERT cells were analyzed by hTR FISH (hTR, red) and TRF1 IF (TRF1, green). Merge panels show superimposition of hTR and TRF1 panels; yellow indicates signal overlap. Some colocalizations of hTR and telomeres are denoted with arrowheads. (B) hTR localizes to most telomeres in VA13+hTR+hTERT cells. hTR FISH (hTR, red) and TRF2 IF (TRF2, green) were performed on VA13 cells that stably express both hTR and hTERT. Arrowheads point to representative examples of hTR and TRF2 colocalizations in each panel. Scale bars, 10 μm.

Figure 7.

Localization of human telomerase RNA (hTR) to Cajal bodies and telomeres is linked to assembly with telomerase reverse transcriptase (hTERT). The simplest model that arises from the current data are that assembly of hTR and hTERT is a prerequisite for the trafficking of telomerase to Cajal bodies and telomeres. It is not clear whether hTR and hTERT assemble before or after localization to Cajal bodies (indicated by question mark). See Discussion for details.

Comment in

Mol Biol Cell. 19:3615.

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Telomerase reverse transcriptase is required for the localization of telomerase RNA to cajal bodies and telomeres in human cancer cells - PubMed (original) (raw)