Telomeric Repeat–Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends

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Science  02 Nov 2007:
Vol. 318, Issue 5851, pp. 798-801
DOI: 10.1126/science.1147182


Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes, are essential for chromosome stability. Until now, telomeres have been considered to be transcriptionally silent. We demonstrate that mammalian telomeres are transcribed into telomeric repeat–containing RNA (TERRA). TERRA molecules are heterogeneous in length, are transcribed from several subtelomeric loci toward chromosome ends, and localize to telomeres. We also show that suppressors with morphogenetic defects in genitalia (SMG) proteins, which are effectors of nonsense-mediated messenger RNA decay, are enriched at telomeres in vivo, negatively regulate TERRA association with chromatin, and protect chromosome ends from telomere loss. Thus, telomeres are actively transcribed into TERRA, and SMG factors represent a molecular link between TERRA regulation and the maintenance of telomere integrity.

Telomeres fulfill essential functions for chromosome stability during mitosis and meiosis, and they equip normal human somatic cells with a cellular clock that determines their replicative lifespan (13). Mammalian telomeres comprise tandem arrays of duplex 5′-TTAGGG-3′ repeats, with the G-rich strand extending beyond its complement to form an overhang (2, 3). The double-stranded part of telomeres is bound to a multiprotein complex known as shelterin, which prevents telomeres from being recognized and processed as double-stranded breaks (4). The heterochromatic state of telomeres, their gene-less nature, and their ability to silence transcription of experimentally inserted subtelomeric reporter genes, which is known as the telomere position effect (TPE) (57), supported the idea that telomeres are transcriptionally silent.

In immortal cells, telomerase promotes telomere length homeostasis by adding telomeric repeats to the 3′ ends of chromosomes. The telomerase holoenzyme comprises a reverse transcriptase catalytic subunit, the telomerase RNA moiety, and several accessory factors (2, 3, 8). The Saccharomyces cerevisiae Est1 protein binds directly to the telomerase RNA and to the single-stranded telomeric DNA binding protein Cdc13p, which recruits telomerase to chromosome ends in S phase (911). Three Est1p-like proteins (EST1A, EST1B, and EST1C) have been identified in humans (12, 13). EST1A and EST1B interact with telomerase, and overexpression of EST1A leads to telomeric fusions and telomere shortening (12, 13). EST1A, EST1B, and EST1C have also been identified as human orthologs of the Caenorhabditis elegans SMG proteins SMG6, SMG5, and SMG7, respectively (1316), which are effectors for nonsense-mediated mRNA decay (NMD) (14). The human NMD core machinery comprises four other SMG polypeptides: UPF1 (SMG2), UPF2 (SMG3), UPF3 (SMG4), and SMG1 (14).

To test whether telomeres are transcriptionally silent, we performed Northern blot analysis of RNA from a human cervical cancer cell line (HeLa) using strand-specific telomeric probes. This approach revealed the existence of TERRA ranging in size from ∼100 bases up to at least 9 kilobases (Fig. 1, A and B). The TERRA signal was abolished upon ribonuclease (RNase) treatment, confirming that it originated from RNA (Fig. 1A). TERRA molecules are found exclusively in nuclear fractions and contain UUAGGG repeats. We only detected a faint signal for the complementary CCCUAA repeats (Fig. 1A), suggesting that CCCUAA-containing RNA molecules might exist only at very low levels. TERRA molecules were detected in different human and rodent cell lines (fig. S1A), indicating conservation of TERRA in mammals.

Fig. 1.

Identification of TERRA. (A) Northern blot analysis of HeLa cytoplasmic (c) and nuclear (n) RNA with strand-specific telomeric and 28S ribosomal RNA (rRNA) probes. Black arrowheads point to nuclear precursors of 28S rRNA (fractionation controls), and the white arrowhead points to the mature 28S rRNA. (B) Northern blot analysis of nuclear RNA with a DNA probe from the Xp/Yp subtelomere. Ethidium bromide (EtBr) staining of the gel is shown as loading control. (C) HeLa nuclear RNA was reverse transcribed (RT) with oTel or with oTR. Control RT reactions were performed by omitting the RT step or by performing the RT with antisense oligonucleotides (oTelαs and oTRαs). PCR was performed with the indicated primer pairs and templates (TMP). gn, genomic DNA. PCR products were visualized by EtBr staining (black arrowheads in upper panels) or by hybridization to an oligonucleotide probe (o2) recognizing a specific subtelomeric region distal to o1 (lower panels).

Hybridization of nuclear RNA blots with a probe derived from the subtelomeric region of human chromosome Xp/Yp generated a TERRA-like hybridization pattern (Fig. 1B). Therefore, human subtelomeres are also transcribed, and TERRA transcription might also start at subtelomeric loci and proceed into the telomeric tract. To test this hypothesis, we reverse transcribed nuclear RNA using a telomeric 5′-(CCCTAA)5-3′ oligonucleotide (oTel) complementary to the putative UUAGGG repeats in TERRA and polymerase chain reaction (PCR)–amplified the derived cDNA using primer pairs located in the subtelomeric regions of chromosomes Xp/Yp, 17p, 11q, and 2p (o1 and o2) (Fig. 1C) (17). The PCR products matched the products stemming from PCR amplifications of genomic DNA (Fig. 1C). Thus, TERRA molecules are transcribed from different telomeres and are composed of subtelomeric-derived RNA and UUAGGG repeats. We also reverse transcribed human nuclear RNA with an oligonucleotide (oTR) containing five 5′-CCCTAA-3′ repeats at the 3′ end and 20 distinctive nucleotides at the 5′ end (oTL) and PCR-amplified the cDNA with the oTL primer in combination with oligonucleotides for subtelomeres of chromosomes 11q and Xp/Yp (o1 in Fig. 1C). The PCR products were heterogeneous in size and positive to Southern hybridization with oligonucleotides (o2 in Fig. 1C) containing sequences that were specific for subtelomeric regions distal to the oligonucleotides used for the PCR. The heterogenous size distribution of the PCR products is inferred to be due to the presence of many UUAGGG repeats in the 3′ region of TERRA and to the annealing of oTR in different registers during reverse transcription.

To analyze the cellular localization of TERRA, we performed RNA fluorescence in situ hybridization (RNA-FISH) experiments. Hybridization of detergent-extracted nuclei with a fluorescently labeled CCCTAA repeat–containing probe revealed an RNase-sensitive punctate nuclear staining: ∼30% of HeLa and primary human lung fibroblasts (HLFs) displayed 3 to 7 TERRA foci, whereas 80 to 100% of human osteosarcoma cells and murine renal cancer cells displayed 20 to 40 foci (Fig. 2A and fig. S1, B and C). Consistent with the Northern blots, our RNA-FISH analysis failed to detect CCCUAA repeat–containing RNA molecules. We then performed RNA-FISH after indirect immunofluorescence, using antibodies against the human shelterin component hRap1 (18). TERRA foci colocalized with Rap1 foci (Fig. 2B and fig. S1D), indicating that TERRA is a component of human telomeric heterochromatin. We also detected TERRA molecules at the ends of metaphase chromosomes from primary mouse embryo fibroblasts (MEFs) (Fig. 2C). The association of TERRA with transcriptionally silent metaphase chromosomes indicates that at least some TERRA remains telomere-associated after its synthesis.

Fig. 2.

Telomeric localization of TERRA. (A) RNA-FISH experiments with strand-specific telomeric DNA probes on HeLa cells. Red and green signals correspond to the probes detecting CCCTAA-CCCUAA repeats or TTAGGG-UUAGGG repeats, respectively. Experiments were performed both in native (to detect RNA) and denaturing (to detect DNA) conditions. 4′,6′-diamidino-2-phenylindole (DAPI)–stained nuclei are in gray. (B) RNA-FISH experiments performed on HLF cells after indirect immunostaining with antibodies to human Rap1. TERRA signals are in red, hRap1 signals are in green, and colocalization of the two signals generates yellow signals in the merged panels. (C) RNA-FISH experiments performed on metaphase chromosomes prepared from MEFs. TERRA signals are in green, and DAPI-stained DNA is in red.

Several SMG proteins are involved in genome stability and telomere maintenance (12, 13, 19, 20). To determine whether SMG proteins regulate TERRA at chromosome ends, we infected HeLa cells with lentiviruses expressing short hairpin RNAs (shRNAs) directed against UPF1, EST1A, SMG1, and UPF2 (fig. S2). Depletion of UPF1, EST1A, or SMG1 induced a substantial increase in the number of cells displaying telomere-associated TERRA foci as well as in the number of foci per positive cell (Fig. 3, A and B, fig. S3, A and B, and fig. S4). A milder increase was observed for UPF2-depleted cells, although the steady-state levels of the NMD substrate BAG1 (21) were similarly increased upon depletion of all tested SMG factors (fig. S3D). The observed accumulation of TERRA in nuclei of SMG-depleted cells was not due to accumulation of cells in different phases of the cell cycle (Fig. 3C and fig. S3C), nor was it the result of substantially increased total TERRA levels (fig. S3D). Furthermore, the approximate half-life for TERRA of 3 hours was not augmented in cells depleted for UPF1 or EST1A (fig. S5). These results suggest that SMG proteins promote the displacement of TERRA from telomeric chromatin and that different SMG proteins might play nonredundant roles in this process. Supporting a direct function for SMG proteins in regulating TERRA at chromosome ends, chromatin fractionation experiments followed by Western blot analysis revealed that all seven SMG proteins are present in chromatin fractions derived from either asynchronous or S-phase HeLa cells (fig. S6A). In addition, chromatin immunoprecipitation experiments with HeLa cell extracts demonstrated that chromatin-bound SMG proteins are several-fold enriched at telomeres in vivo over Alu-repeat sequences (fig. S6, B and C).

Fig. 3.

SMG proteins promote TERRA dissociation from chromatin. (A) HeLa cells were infected with lentiviruses expressing shRNAs directed against the indicated SMG proteins or empty vector (shEV), and RNA-FISH experiments were performed to detect chromatin-associated TERRA (in red). Numbers indicate the counted TERRA foci for each nucleus. (B) Classification of infected cells according to the number of TERRA foci. 100 to 200 cells were counted for each infection. Means (bars) and SDs (error bars) are from two independent experiments. (C) Fluorescence-activated cell sorting analysis of the same cells as in (A) and (B). PI, propidium iodide.

We then transfected HeLa cells with plasmids expressing shRNAs against different SMG proteins, which led to more efficient depletion than the lentiviral-mediated depletion used above (fig. S2). Terminal restriction fragment (TRF) analysis in UPF1- and EST1A-depleted cells did not reveal noticeable changes in TRF lengths. Nevertheless, a decrease in the overall intensity of the telomeric smear was seen when the telomeric signal obtained from chromosome ends was normalized to the signal derived from interstitial telomeric repeats (Fig. 4A). Thus, UPF1 and EST1A depletion might lead to the sudden loss of entire telomeric tracts at some chromosome ends. Consistently, DNA-FISH analysis of metaphase chromosomes from UPF1- or EST1A-depleted HeLa cells revealed a five- to sevenfold increase in telomere-free chromosome ends. A twofold increase in associated sister telomeres and a four- to fivefold increase in telomeric fragments were also observed (Fig. 4, B and C, and fig. S7). Similar results were obtained for SMG1-depleted cells, although telomere rearrangements were observed at lower frequencies in cells depleted for UPF2 or EST1C (Fig. 4C and fig. S7). Because plasmid shRNA–mediated depletion of UPF1, EST1A, and UPF2 led to similar stabilization of the NMD reporter β-globin NS39 RNA (22) (fig. S2), at least a part of the observed telomeric defects is not an indirect consequence of loss of canonical NMD. Consistently, RNA microarray analysis of cells depleted for UPF1 did not disclose pronounced deregulation of transcripts coding for known telomeric factors, including the six shelterin components (23). Finally, scoring of metaphases depleted for UPF1, EST1A, and SMG1 revealed accumulation of chromosome and chromatid breaks at subtelomeric and intrachromosomal locations (Fig. 4B), which is consistent with more general roles for UPF1 and SMG1 in genome stability (19, 20) and suggestive of similar roles for EST1A.

Fig. 4.

SMG proteins protect human cells from loss of entire telomere tracts. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with plasmids expressing shRNAs against UPF1 and EST1A or with empty vector controls. Numbers at the bottom indicate the ratio between the signal associated to the bulk of telomeres and the signal associated to intrachromosomal telomeric repeats (ITs), relative to shEV-transfected cells. Means and SDs are from three independent experiments. (B) Complete metaphases and enlarged examples from telomeric DNA–FISH experiments performed on metaphase chromosomes from HeLa cells treated as in (A). Telomeric sequences are in green, and DAPI-stained chromosomes are in red. Arrowheads point to telomere-free chromosome ends (TFEs). Asterisks indicate telomeric fragments (TFs), associated sister telomeres (ASTs), a subtelomeric chromatid break (Sub), and an intrachromosomal chromatid break (Br). (C) Frequencies of telomeric aberrations observed in SMG-depleted cells.

Mammalian telomeres have genelike properties in that they are actively transcribed into TERRA molecules. Artificially induced strong transcription of a budding yeast telomere alleviated the TPE exerted by the transcribed telomere and provoked telomere shortening (24), suggesting a role for telomeric RNA in organizing telomere architecture. It is possible that TERRA promotes telomeric heterochromatin assembly by mechanisms similar to the X-chromosome inactivation in females, which is mediated by the long noncoding Xist RNA (25), or to the RNA interference–mediated heterochromatinization of the telomeric dh-homologous region in fission yeast (26). SMG proteins regulate TERRA at chromosome ends, and UPF1 and EST1A physically interact with DNA polymerase δ and telomerase, respectively (12, 13, 19, 27). We speculate that the telomeric defects induced by SMG-depletion could derive from the loss of coordination between TERRA and key enzymatic activities that assure telomere replication and length homeostasis.

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Materials and Methods

Figs. S1 to S7

Tables S1 and S2


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