Cell cycle-regulated trafficking of human telomerase to telomeres

Abstract

Telomerase synthesizes telomeres at the ends of human chromosomes during S phase. The results presented here suggest that telomerase activity may be regulated by intranuclear trafficking of the key components of the enzyme in human cells. We examined the subcellular localization of endogenous human telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT) in HeLa cervical carcinoma cells. Throughout most of the cell cycle, we found that the two essential components of telomerase accumulate at intranuclear sites separate from telomeres. However, during S phase, both hTR and hTERT are specifically recruited to subsets of telomeres. The localization of telomerase to telomeres is dynamic, peaking at mid-S phase. We also found complex associations of both hTR and hTERT with nucleoli and Cajal bodies during S phase, implicating both structures in the biogenesis and trafficking of telomerase. Our results mark the first observation of human telomerase at telomeres and provide a mechanism for the cell cycle-dependent regulation of telomere synthesis in human cells.

Figure 1.

Localization of human telomerase RNA in HeLa cells at various stages of the cell cycle. hTR (red, detected by FISH) and coilin (green, Cajal body marker protein detected by IF) were analyzed by fluorescence microscopy in cells 0 (G₁/S), 4 (mid-S), 8 (G₂), 10 (M), 12 (early G₁), and 15 (mid-G₁) hours after release from double thymidine block. Differential interference contrast (DIC) panels show differential interference light microscopy data. DAPI panels shows DNA staining. Merge panels show superimposition of hTR and coilin fluorescence data (yellow indicates overlap of signal). Arrows in mid-S merge panel indicate hTR foci that do not colocalize with coilin.

Figure 2.

Human telomerase RNA is found at the periphery of nucleoli and at telomeres during S phase. (A) hTR and coilin seem to be associated with the surface of nucleoli during early S phase. The localization of hTR (red, detected by FISH) is superimposed on coilin (green, detected by IF, 1) or cellular architecture (visible by DIC light microscopy, 2 and 4) in indicated panels. The cells shown are in early S phase. Arrowheads denote nucleoli exhibiting apparent peripheral or surface hTR signals. (B) hTR associates with telomeres during mid-S phase. hTR (red, detected by FISH) and telomeres (green, detected by TRF1 IF or telomere FISH as indicated) were analyzed in mid-S-phase cells. DAPI panel shows DNA staining. BrdU panel shows a mid-S-phase pattern. Arrows in merge panels indicate foci where both hTR and telomeres are present.

Figure 3.

Cellular localization of human telomerase reverse transcriptase. (A) Intranuclear foci recognized by hTERT antibody 2C4 are specific to hTERT. The panels show 2C4 immunostaining of HeLa cells and HeLa cells within which hTERT was knocked down by RNA interference (-hTERT) and of IMR90 primary fibroblasts and an IMR90 strain that overexpresses hTERT (+hTERT). Data in each pair of panels were normalized to allow visual comparison. (B) hTERT is found in nucleoli in early S-phase cells. The localization of hTERT (red, detected by IF) is superimposed on cellular architecture visible by DIC light microscopy in merge panels. (C) hTERT is found at telomeres in mid-S-phase cells. hTERT (red) and TRF1 (green) localization is shown. Arrowheads indicate representative foci where both hTERT and TRF1 are present.

Figure 4.

Temporal patterns of association of human telomerase RNA and telomerase reverse transcriptase with nuclear structures during S phase in HeLa cells. The percentage of cells in which hTR (A) and hTERT (B) was found associated with each structure in G₁/S, early S, mid-S, late S, and S/G₂ phase cells is indicated as follows: telomeres (•) and nucleoli (hTR peripheral and hTERT internal) (▴) on left axis; colocalization with Cajal bodies (♦) and Cajal body-associated foci (▪) on right axis (note difference in scale of axes). Each point plotted represents an average of at least 158 (and as many as 698) total cells counted from at least two (and as many as 6) experiments, with the exception of the S/G₂ time point for hTERT, which represents data from a single experiment.

Figure 5.

Human telomerase RNA and telomerase reverse transcriptase are found in foci adjacent to Cajal bodies during S phase. Coanalysis of coilin (blue) and hTR (red) or hTERT (red) in early or mid-S-phase HeLa cells is shown. Merge panels show superimposition of coilin and hTR or hTERT signals. Insets show enlargements of close associations of distinct coilin and hTR or hTERT foci (indicated by arrowheads in merge panel).

Figure 6.

Model for cell cycle-regulated trafficking of telomerase subunits to telomeres during S phase. The predominant phase-specific localization of hTR and hTERT is shown for G₁ (A), early S (B), and mid-S (C). The arrows indicate possible trafficking pathways accounting for the observed localizations. See text for details.