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. 2004 Mar 1;164(5):647-52.
doi: 10.1083/jcb.200310138. Epub 2004 Feb 23.

Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal

Beáta E Jády  1 , Edouard Bertrand, Tamás Kiss
Affiliations

Human telomerase RNA and box H/ACA scaRNAs share a common Cajal body-specific localization signal

Beáta E Jády et al. J Cell Biol. .

Abstract

Telomerase is a ribonucleoprotein reverse transcriptase that uses its RNA component as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes. Here, fluorescence in situ hybridization demonstrates that in HeLa cancer cells, human telomerase RNA (hTR) accumulates in the nucleoplasmic Cajal bodies (CBs). Localization of transiently expressed hTR to CBs is supported by a short sequence motif (411-UGAG-414) that is located in the 3'-terminal box H/ACA RNA-like domain of hTR and that is structurally and functionally indistinguishable from the CB-specific localization signal of box H/ACA small CB-specific RNAs. In synchronized HeLa cells, hTR shows the most efficient accumulation in CBs during S phase, when telomeres are most likely synthesized. CBs may function in post-transcriptional maturation (e.g., cap hypermethylation of hTR), but they may also play a role in the assembly and/or function of telomerase holoenzyme.

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Figures

Figure 1.
Figure 1.
Schematic structure of hTR. The template region and the H and ACA boxes are indicated. The sequence of the conserved region 7 (CR7) is shown, and the putative CAB box motif is boxed.
Figure 2.
Figure 2.
In situ localization of endogenous hTR in HeLa cells. HeLa cells were hybridized with a mixture of three fluorescent oligonucleotide probes complementary to hTR from G135 to A174, A346 to A382, and U416 to A449. The U85 scaRNA was detected with an oligonucleotide probe complementary to U85 from U13 to G53. CBs were stained with antibodies directed against p80-coilin or SMN. Nucleoli were visualized by transient expression of GFP-fibrillarin (Dundr et al., 2000). Nuclei were stained with DAPI. Unless indicated otherwise, bars represent 10 μm.
Figure 3.
Figure 3.
CAB box–dependent localization of hTR to CBs. Schematic structure of the hTR gene transfected into HeLa cells is shown. Sequences of the CAB box motifs of the wild-type (hTR) and mutant (hTR-m1 and hTR-m2) RNAs are shown. Altered nucleotides are indicated by lowercase letters. Cells were hybridized with a fluorescent probe complementary to hTR from A346 to A382. CBs were stained with an antibody against p80-coilin. Expression of GFP-fibrillarin visualized both nucleoli and CBs. Nuclei of nontransfected cells are circled. Bars, 10 μm.
Figure 4.
Figure 4.
Cell cycle–dependent accumulation of hTR in CBs. (A) FISH. Human HeLa cells were synchronized by the double-thymidine blocking procedure. Progression of synchronized cells through cell cycle was monitored by FACS® analysis. The results of FACS® analysis calculated with CELLQuest software (Becton Dickinson) are indicated on the right. Each specimen was double stained with a mixture of hTR-specific fluorescent oligonucleotides and an anti-p80-coilin antibody. Bars, 10 μm. (B) Quantification of hTR accumulation in CBs. To estimate the relative amount of hTR accumulating within CBs in different cell cycle phases, pictures of the endogenous hTR localization taken under identical conditions were analyzed with MetaMorph® software (Universal Imaging Corp.). For each CB, the difference between the maximum relative grayscale value of pixels representing the CB and the average relative grayscale value of pixels representing the surrounding nucleoplasm was determined. For each cell cycle phase, the obtained values for 100 randomly selected CBs were averaged. To define the relative value of fluorescence, the intensity of early S phase CBs was taken as 100%. Statistical analysis (Friedman's test and Tukey multiple comparisons test) confirmed that the differences between the measures obtained for S, G2, and G1 cells are significant at the 0.001 significance level. (C) Northern analysis. RNAs extracted from nonsynchronized (control) or synchronized HeLa cells in the early S, late S, G2, G1, and G1/S transition phases. 10-μg RNA samples were analyzed by Northern blotting with a mixture of labeled oligonucleotides complementary to the U85 scaRNA and hTR. Lane M, size markers.
Figure 5.
Figure 5.
hTR carries a TMG cap. HeLa cellular RNAs immunoprecipitated with monoclonal (H-20) and polyclonal (α-TMG; provided by R. Lührmann, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany) antibodies against TMG were mapped by RNase A/T1 protection using antisense RNA probes complementary to hTR, U2, and U19. Control mappings performed with Escherichia coli tRNA (control), HeLa total RNA (total), or RNA precipitated. with a nonimmune serum (nonimm) are also shown. Lane M, size markers.
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