|TERRA: telomeric repeat-containing RNA.|
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|PMID: 19629047 Owner: NLM Status: MEDLINE|
|Telomeres, the physical ends of eukaryotic chromosomes, consist of tandem arrays of short DNA repeats and a large set of specialized proteins. A recent analysis has identified telomeric repeat-containing RNA (TERRA), a large non-coding RNA in animals and fungi, which forms an integral component of telomeric heterochromatin. TERRA transcription occurs at most or all chromosome ends and it is regulated by RNA surveillance factors and in response to changes in telomere length. TERRA functions that are emerging suggest important roles in the regulation of telomerase and in orchestrating chromatin remodelling throughout development and cellular differentiation. The accumulation of TERRA at telomeres can also interfere with telomere replication, leading to a sudden loss of telomere tracts. Such a phenotype can be observed upon impairment of the RNA surveillance machinery or in cells from ICF (Immunodeficiency, Centromeric region instability, Facial anomalies) patients, in which TERRA is upregulated because of DNA methylation defects in the subtelomeric region. Thus, TERRA may mediate several crucial functions at the telomeres, a region of the genome that had been considered to be transcriptionally silent.|
|Brian Luke; Joachim Lingner|
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|Type: Journal Article; Research Support, Non-U.S. Gov't; Review Date: 2009-07-23|
|Title: The EMBO journal Volume: 28 ISSN: 1460-2075 ISO Abbreviation: EMBO J. Publication Date: 2009 Sep|
|Created Date: 2009-09-02 Completed Date: 2009-10-22 Revised Date: 2013-06-02|
Medline Journal Info:
|Nlm Unique ID: 8208664 Medline TA: EMBO J Country: England|
|Languages: eng Pagination: 2503-10 Citation Subset: IM|
|EPFL-Ecole Polytechnique Fédérale de Lausanne, ISREC-Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland.|
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Chromatin Assembly and Disassembly
Heterochromatin / metabolism
RNA, Untranslated / chemistry, physiology*
Repetitive Sequences, Nucleic Acid
Telomere / chemistry*
|0/Heterochromatin; 0/RNA, Untranslated|
Journal ID (nlm-ta): EMBO J
Publisher: Nature Publishing Group
Copyright ? 2009, European Molecular Biology Organization
open-access: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation or the creation of derivative works without specific permission.
Received Day: 27 Month: 04 Year: 2009
Accepted Day: 25 Month: 05 Year: 2009
Print publication date: Day: 2 Month: 09 Year: 2009
Electronic publication date: Day: 23 Month: 07 Year: 2009
Volume: 28 Issue: 17
First Page: 2503 Last Page: 2510
Publisher Item Identifier: emboj2009166
PubMed Id: 19629047
|TERRA: telomeric repeat-containing RNA Alternate Title:TERRA and telomeres|
1EPFL-Ecole Polytechnique F?d?rale de Lausanne, ISREC-Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland
2?Frontiers in Genetics' National Center for Competence in Research (NCCR), Geneva, Switzerland
|aEPFL, School of Life Sciences-ISREC, B?timent SV, Station 19, Lausanne 1015, Switzerland. Tel.: +41 21 693 0721; Fax: +41 21 693 0720; E-mail: firstname.lastname@example.org
Telomeres are critical at the cellular level for genome stability, whereas at the organismal level, they act as tumour suppressors and may contribute to ageing. The following roles of telomeres are well established: First, telomeres can regulate the lifespan of cells (Harley et al, 1990; Allsopp et al, 1992; Levy et al, 1992; Bodnar et al, 1998). Telomere shortening occurs at the distal ends because of a combination of nucleolytic processing of chromosome ends and the end replication problem. This shortening can be counteracted by the cellular reverse-transcriptase telomerase, which uses an internal RNA moiety as a template for the synthesis of telomere repeats (Cech, 2004; Blackburn et al, 2006). Telomerase is regulated at individual chromosome ends through telomere-binding proteins to mediate telomere length homoeostasis; however, in humans, telomerase is expressed in most tissues only during the first weeks of embryogenesis (Ulaner and Giudice, 1997). Repression of telomerase in somatic cells is thought to result in a powerful tumour-suppressive function. Short telomeres that accumulate following an excessive number of cell division cycles induce cellular senescence, and this counteracts the growth of pre-malignant lesions. This growth barrier is overcome in most cancers that contain cells that re-express telomerase. Second, telomeres protect natural chromosome ends from unwanted DNA repair activities, which include non-homologous end joining and homologous recombination (Palm and de Lange, 2008). Several telomere-associated proteins are critical for this function. Third, telomeres establish a heterochromatic state at chromosome ends, which is characterized by the presence of trimethylated lysines at position 9 in histone H3 and at position 20 in histone H4, histone hypoacetylation, the accumulation of several isoforms of heterochromatin protein 1 and the hypermethylation of cytosines in CpG dinucleotides that are present in subtelomeric regions (Blasco, 2007; Ottaviani et al, 2008). The heterochromatic state of telomeres may contribute to chromosome positioning and movement within the nucleus and also to the regulation of telomerase. Abnormal telomeric chromatin states have been linked to severe stochastic telomere sequence loss phenotypes, suggesting crucial roles of this structure during telomere replication (Michishita et al, 2008; Yehezkel et al, 2008).
Several telomere-binding proteins that are crucial for the distinct telomere functions outlined above have been identified. These include the six-polypeptide-containing shelterin complex (Palm and de Lange, 2008), which is abundant at telomeres as well as other more rare telomeric proteins. However, relatively little is known mechanistically about which telomere transitions occur between different states for example: when to activate telomerase in a length-dependent and cell-cycle-restricted manner; when to induce cellular senescence when telomeres are very short; and when to lose protection from end fusion upon further erosion. Recent analysis has identified telomeric repeat-containing RNA (TERRA), a large non-coding RNA in animals and fungi, which forms an integral component of telomeric heterochromatin (Azzalin et al, 2007; Luke et al, 2008; Schoeftner and Blasco, 2008). Earlier reports also indicate the existence of telomere transcription in trypanosomes, birds and Diptera (Morcillo et al, 1988; Rudenko and Van der Ploeg, 1989; Solovei et al, 1994). The finding of ribonucleic acids at telomeres adds a new dimension of how functions at the telomere may be orchestrated and regulated. Here, we review recent progress in the understanding of TERRA biogenesis, turnover and function.
TERRA is a heterogeneous non-coding RNA that consists of a combination of subtelomeric and telomeric sequences. To date, all individual telomeres tested in mammals can and do produce TERRA transcripts and TERRA is expressed in most tissues (Azzalin et al, 2007; Azzalin and Lingner, 2008; Schoeftner and Blasco, 2008). TERRA is transcribed in a centromere to telomere direction, indicating that the transcription start site lies within the subtelomeric sequence. In the yeast, Saccharomyces cerevisiae, it has been determined that TERRA transcription occurs at both, telomeres containing or lacking the Y? element. The Y? element is a conserved repeat sequence, present at ?50% of all telomeres in S. cerevisiae. The 5?-end of TERRA derived from telomeres containing the Y? element is relatively homogenous, supporting the idea of a defined start site (Luke et al, 2008). This suggests that the heterogeneity of TERRA stems from its 3?-end, and indicates either that transcription can terminate in multiple places within the telomeric tract, or that TERRA 3?-ends are differentially processed. TERRA is transcribed largely by RNA polymerase II (RNAPII). This conclusion was reached because of the fact that RNAPII was found associated with telomeric DNA through chromatin immunoprecipitation (ChIP) experiments in human, mouse and yeast cells (Luke et al, 2008; Schoeftner and Blasco, 2008). Furthermore, the specific inhibition of RNAPII by ?-amanitin reduces the abundance of TERRA in both human and mouse cells (Schoeftner and Blasco, 2008). Moreover, a temperature-sensitive RNAPII allele (rpb3-2) prevents the accumulation of TERRA in budding yeast (Luke et al, 2008). Although ?-amanitin does reduce, to a large extent, the abundance of TERRA in mammalian cells, it is far less efficient in doing so than the general transcriptional inhibitor, actinomycin D. Where actinomycin D treatment resulted in a 50% reduction in TERRA levels within 2.5?3 h (Azzalin et al, 2007; Schoeftner and Blasco, 2008), only ?30% of TERRA was degraded after 6 h of ?-amanitin treatment, and 20% of initial TERRA was still remaining after 12 h of treatment (Schoeftner and Blasco, 2008). Similar results were seen in mouse cells treated with ?-amanitin, in which TERRA levels never decreased below 50% of initial levels even after 12 h of RNAPII inhibition (Schoeftner and Blasco, 2008). These results indicate that although RNAPII has a major function in promoting TERRA transcription, there may be another RNA polymerase (either RNAPI and RNAPIII) that could potentially have a function in telomeric transcription in mammals. The idea of another RNA polymerase being involved in generation of TERRA would be consistent with recent data in which several subunits of RNAPI and RNAPIII (RPABC1, RPAC1, RPA49, RPA2 and RPA1) were found enriched in formaldehyde-crosslinked and partially purified human telomere fractions (Dejardin and Kingston, 2009). Furthermore in trypanosomatida, telomeres can be transcribed both by RNAPII and, in the subset of trypanosomes that show antigenic variation, by the ?-amanitin-resistant RNAPI (Rudenko and Van der Ploeg, 1989).
Similar to most products of RNAPII transcription, TERRA is polyadenylated at its 3?-end (Azzalin and Lingner, 2008; Luke et al, 2008; Schoeftner and Blasco, 2008). Approximately 7% of human TERRA is polyadenylated (Azzalin and Lingner, 2008), whereas most or all yeast TERRA molecules carry a poly(A) tail (Luke et al, 2008). It is unknown whether TERRA polyadenylation involves the 3?-end cleavage of a longer TERRA precursor or whether terminated transcripts are directly polyadenylated. A canonical 5?-AAUAAA-3? cleavage and a polyadenylation signal is not present at mammalian TERRA 3?-ends, whereas the telomere-derived GU-rich TERRA 3?-ends in yeast bear some resemblance to the canonical U-rich 3?-end-processing signals in this organism. In yeast, the canonical polyadenylation polymerase, Pap1, is responsible for poly(A) addition and it most likely contributes to the stability of the RNA, because TERRA becomes completely destabilized in pap1-1 temperature-sensitive mutants and polyadenylated TERRA is no longer detectable (Luke et al, 2008). How the 5?-end of TERRA is modified has not yet been addressed. If it contains a 7-methylguanosine (m7G) cap structure as do mRNAs, it likely gets uncapped in S. cerevisiae at the time when it becomes a target for Rat1-dependent degradation, which recognizes 5?-uncapped mono-phosphate structures (see below).
Although the specific transcription factors that are responsible for the transcriptional regulation of TERRA have not yet been elucidated, there are indications that some of the telomere-bound proteins that make up the protective cap of telomeres, along with the heterochomatic markers present at telomeres, are involved in controlling TERRA levels. As discussed in the Introduction section, telomeres are often in a heterochromatic state (Blasco, 2007; Ottaviani et al, 2008). Trichostatin A, a histone deacetylase inhibitor, results in increased TERRA levels in human cells (Azzalin and Lingner, 2008) as do mutations in the histone methyltransferases, SUV39H1/2 and SUV4-20H1/2, in mouse cells (Schoeftner and Blasco, 2008) and mutations in the DNA methyltransferase 3b (DNMT3B) in human cells (Yehezkel et al, 2008). These results indicate that TERRA levels are partially controlled through subtle changes in the local heterochromatin, although indirect effects could not be distinguished in these experiments.
Shelterin is a complex composed of six proteins (TRF1, TRF2, Rap1, TIN2, TPP1 and POT1), which binds to and protects telomeres (Palm and de Lange, 2008). Interestingly, TRF1 can interact with RNAPII as shown through co-immunoprecipitation experiments (Schoeftner and Blasco, 2008). It has been reported that when TRF1 was depleted using small-interfering RNA (siRNA), overall levels of TERRA decreased twofold, which suggests that TRF1 supports TERRA transcription (Schoeftner and Blasco, 2008). It is unlikely, however, that TRF1 acts as a conventional transcriptional activator. First, depletion of TRF1 does not result in a corresponding loss of RNAPII associated with telomeric DNA. Second, TRF1 is restricted to the telomeric tract and likely does not spread into the subtelomere, wherein transcription is presumed to initiate. Finally, the overproduction of TRF1 results in less TERRA production, rather than more, although it should be noted that telomeres shorten in length upon TRF1 overexpression and this may also influence TERRA transcription (Benetti et al, 2008; Schoeftner and Blasco, 2008; Munoz et al, 2009). Overall, these data suggest that TRF1 promotes RNAPII progression through the telomeric tract, but it does not directly influence RNAPII recruitment (see Figure 1, TERRA transcription).
It is important to re-visit all of the known telomere-associated proteins (shelterin and non-shelterin components) and re-assess their functions in terms of TERRA regulation. This also includes several proteins that could be potentially involved in transcription elongation and regulation, which were detected in partially purified telomere fractions (Dejardin and Kingston, 2009).
TERRA is exclusively found in nuclear RNA fractions from human and mouse cells, and is likely restricted to the nucleus in yeast as well (see below). Furthermore, TERRA can be detected using RNA fluorescence in situ hybridization (RNA-FISH) at a subset of telomeres in interphase cells, and it is present on human and mouse chromosome ends in the metaphase of the cell cycle (Azzalin et al, 2007; Schoeftner and Blasco, 2008). The fact that TERRA associates with telomeres suggests that TERRA may be an integral part of the telomere and could potentially be important for structural integrity. However, because TERRA is detected by FISH in only a subset of telomeres, it seems more likely that TERRA is not an essential or a permanent constituent of the telomeric chromatin, but rather has a transient structural function during telomere assembly or it may have regulatory roles in a subset of telomeres that may correspond to a specific functional state. A telomere autonomous regulation would be compatible with telomeric-state-dependent functions; for example, in the establishment, but not necessarily the maintenance, of telomeric heterochromatin or in the regulation of telomerase in cis in a length-dependent manner. Although TERRA remains associated with telomeres, it has not been shown whether TERRA molecules can move from one telomere to another or whether the RNA remains associated with the telomere that it was transcribed from.
There are two potential means by which TERRA could be tethered to telomeres. One possibility would be through interactions of a telomeric protein with the RNA or, alternatively, through a direct interaction of TERRA with the telomeric DNA. Both modes of association are possible and are indeed not mutually exclusive. In yeast, mainly indirect evidence has hinted that DNA?RNA hybrid formation is occurring to some extent. It was shown that overexpression of RNaseH, which degrades the RNA moiety in a DNA?RNA hybrid, could specifically reduce TERRA levels in a genetic background in which TERRA accumulates (Luke et al, 2008). Furthermore, it was shown in vitro that telomeric DNA and telomeric RNA together form an intermolecular heat-stable G-quadruplex structure (Xu et al, 2008). To date, there is no evidence that telomeric proteins are involved in the tethering of TERRA to chromosome ends. POT1, a good candidate that binds to telomeric ssDNA in mammals, does not interact with TERRA in vitro (S Redon and JL, unpublished data). Another candidate in mammalian cells is hnRNP A1, an abundant RNA-binding protein that binds specifically to UAGGGA/U repeats in vitro (Burd and Dreyfuss, 1994). UUAGGG repeats are very abundant in the portion of mammalian TERRA that is transcribed from telomeric repeats. hnRNP A1 has also been implicated in telomere length control (LaBranche et al, 1998) and it binds to single-stranded telomeric repeats in vitro (McKay and Cooke, 1992) and to telomeres in vivo (Zhang et al, 2006). Thus, it might function as a molecular bridge between telomeric DNA and TERRA. Telomere purification also showed other RNA-binding proteins, including FUS, RNA-binding protein 14, cold-induced RNA-binding protein and RNA-binding protein 8A (Dejardin and Kingston, 2009), all of which might be involved in TERRA localization to telomeres (Figure 1, see TERRA telomere association).
Several negative regulators of both TERRA stability and TERRA localization have now been documented. The nonsense-mediated RNA decay (NMD) machinery, which is best known for its role in the degradation of mRNAs containing pre-mature termination codons, has also an important function in the displacement of TERRA from human telomeres (Azzalin et al, 2007; Chawla and Azzalin, 2008). From the key players in the NMD pathway, UPF1, SMG1 and EST1A/SMG6 were shown to regulate TERRA at telomeres, as their depletion by RNA interference (RNAi) led to a several-fold increase of telomeres with TERRA foci. However, neither total levels of TERRA nor TERRA half-life was affected by the depletion of these factors. NMD proteins may interact directly with TERRA at telomeres because NMD proteins can be detected at telomeres in vivo by ChIP (Azzalin et al, 2007). Furthermore, the NMD factor, EST1A/SMG6, physically interacts with telomerase (Reichenbach et al, 2003; Snow et al, 2003; Redon et al, 2007). Two possible scenarios on how NMD factors diminish TERRA abundance at telomeres can be envisioned. First, UPF1/SMG1/EST1A may be required only for the displacement of TERRA without influencing the rate of TERRA degradation. Second, UPF1/SMG1/EST1A may be involved in TERRA degradation, but only locally at the telomere, and such small changes in TERRA levels are not discernable on northern blots. In this respect, it is interesting to note that EST1A/SMG6 contains a PIN domain at its C terminus, which shows RNA endonuclease activity during NMD (Huntzinger et al, 2008; Eberle et al, 2009). Distinguishing between these two possibilities will be technically challenging.
It is not known whether yeast TERRA resides at the telomere as is the case in complex eukaryotes; therefore, regulators of TERRA localization have not yet been addressed in this organism. However, it is intriguing that inactivation of the NMD machinery in S. cerevisiae leads to shorter telomeres, although it had been proposed that this effect was due to increased expression levels of the telomere-capping factors, Stn1 and Ten1, which were observed in NMD mutants (Dahlseid et al, 2003; Enomoto et al, 2004). In yeast, NMD mutants did not affect overall TERRA levels (Luke et al, 2008), but similar to human cells, they might only locally affect TERRA, rendering the effect undetectable on northern blots.
It has been determined, however, that the stability of TERRA in yeast is tightly regulated by the 5?- to 3?-exonuclease, Rat1. In wild-type cells, TERRA is almost undetectable using standard northern blotting procedures, but accumulates significantly when Rat1 is inactive. These effects are likely to be direct. Mutation in the RAT1 gene increases the half-life of TERRA; moreover, Rat1 can be found to be associated with telomeres through ChIP experiments. On the other hand, Xrn1, the cytoplasmic paralog of Rat1, has no effect on TERRA levels, supporting the notion that, similar to its human counterpart, yeast TERRA is nuclear. Trf4, an alternative poly(A) polymerase that is often associated with exosomal-mediated degradation, has no effect on TERRA levels when mutated singly; however; when combined with a Rat1 mutation, the double-mutant shows a dramatic increase in TERRA levels. This suggests that Trf4 has a minor function in TERRA degradation on its own; however, when Rat1 is mutated and TERRA levels are higher, the contribution of Trf4 becomes more noticeable. In this respect, analysis of double mutants (with one mutation being the rat1-1 mutation) may be instrumental in finding additional regulators of TERRA stability and, furthermore, of TERRA transcriptional activation (Figure 1, see TERRA removal and degradation).
As discussed above, in human cells, TERRA is displaced or degraded at telomeres by NMD factors that physically interact with telomeric chromatin (Azzalin et al, 2007). Among these factors, EST1A/SMG6 was also identified through its sequence similarity with the S. cerevisiae telomerase-associated protein, Est1 (Reichenbach et al, 2003; Snow et al, 2003). Moreover, similar to yeast Est1, human EST1A/SMG6 physically interacts with telomerase (Reichenbach et al, 2003; Snow et al, 2003; Redon et al, 2007). The S. cerevisiae Est1 protein binds directly to telomerase RNA and to the single-stranded telomeric DNA-binding protein, Cdc13p, to recruit telomerase to chromosome ends in the S phase. Therefore, in est1? cells, telomerase cannot act and telomeres shorten (Lundblad and Szostak, 1989; Pennock et al, 2001; Chan et al, 2008). The recruitment and activation of telomerase at chromosome ends is not well understood in complex eukaryotes, including humans, and the role of EST1A/SMG6 in this process is uncertain. The association of EST1A/SMG6 with telomerase is compatible with a role in telomerase regulation, but its effects on TERRA displacement at telomeres suggest that EST1A/SMG6 may regulate telomerase through TERRA. Indeed, the TERRA-mimicking RNA oligonucleotide (UUAGGG)3 inhibits telomerase activity in vitro as determined in the TRAP assay (Schoeftner and Blasco, 2008), and because TERRA is more abundant when telomeres are long (Azzalin et al, 2007; Schoeftner and Blasco, 2008), it has been proposed that telomerase may be regulated by TERRA in a telomere-length-dependent manner (Schoeftner and Blasco, 2008). Consistent with a negative regulatory role of TERRA for telomerase is also the finding that in several tumours, lower levels of TERRA were detected than in the corresponding normal tissue (Schoeftner and Blasco, 2008). Furthermore, in tumour-derived and in in vitro-immortalized cell lines, the presence of telomerase activity correlates with lower TERRA levels when compared with cell lines that use the recombination-based ALT pathway to maintain telomeric ends (Ng et al, 2009). Genetic experiments in S. cerevisiae provide more conclusive evidence that TERRA indeed regulates telomerase in vivo. In the rat1-1 mutant background, TERRA is upregulated and telomeres are shorter than in wild-type cells because of an impairment in telomerase-mediated telomere elongation (Luke et al, 2008). The overexpression of RNaseH reduced TERRA levels and could overcome the short telomere phenotype, indicating that a DNA/TERRA hybrid was responsible for the effect. Further support for the role of TERRA in inhibiting telomerase in vivo stems from an observation that forced telomere transcription (through the use of a strong Gal promoter) leads to telomere shortening of the transcribed telomere in cis (Sandell et al, 1994). It will be interesting to elucidate the mode of inhibition of the enzymatic activity of telomerase by TERRA and to determine its possible effects on the recruitment of telomerase to telomeres.
Large ncRNAs can mediate epigenetic changes by recruiting chromatin-remodelling complexes to specific genomic loci. For example, in the human HOXD locus, the 2.2-kb HOTAIR ncRNA interacts with the polycomb repressive complex 2, resulting in trimethylated histone H3 at lysine 27 (H3K27) (Rinn et al, 2007). Similarly, for X chromosome inactivation of one of the two X chromosomes in females, transcription of the 1.6-kb RepA RNA within the Xist locus is involved in recruiting polycomb group proteins for H3K27 trimethylation (Zhao et al, 2008). Thus, it would not be surprising if TERRA also functions as a recruiter for chromatin-modifying enzymes at telomeres to establish or stabilize the heterochromatic state.
More clearly established is another parallel between Xist and TERRA, in which both are regulated by SMG proteins. Female mouse embryonic stem (ES) cell lines depleted for RENT1 (the murine ortholog of UPF1) or for UPF2 fail to form Xist RNA domains upon differentiation, and subsequently fail to undergo X-inactivation (Ciaudo et al, 2006). Thus, nuclear SMG proteins may contribute to chromatin organization at several loci that involve long ncRNAs. However, methods that allow a direct manipulation of TERRA transcription or stability will be required to elucidate this issue. Furthermore, TERRA has been shown to colocalize with Xist (Schoeftner and Blasco, 2008; Zhang et al, 2009) and specifically with the distal telomere of the inactive X chromosome in female mouse embryo fibroblasts (Zhang et al, 2009). Curiously, TERRA localization to the inactive X does not depend on Xist expression, which also indicates that TERRA is not dependent on X chromosome inactivation but rather likely plays a telomere-specific heterochromatinization role. In the fission yeast Schizosaccharomyces pombe, siRNAs are produced from subtelomeric repetitive elements referred to as dh repeats, whereby both the RNAi RITS (RNA-induced initiation of transcriptional silencing) pathway and the major telomere-binding protein Taz1 (ortholog of human TRF1 and TRF2) establish Swi6 (ortholog of HP1) heterochromatin at telomeres in parallel pathways (Kanoh et al, 2005). Two lines of evidence suggest a possible function for RNAi in maintaining the telomeric structure and TERRA regulation in mammals. First, upon transfection of mouse cells with synthetic siRNAs, irrespective of the siRNA sequence used, the Argonaute 1 protein associates with telomeres and TERRA expression increases (Ho et al, 2008). Second, the inactivation of Dicer had effects on TERRA levels in mouse ES cells in two different studies, although it needs to be resolved whether levels increase or decrease, as the reports were conflicting on this issue (Schoeftner and Blasco, 2008; Zhang et al, 2009). It will be interesting to determine whether TERRA also base pairs with other cellular RNAs and whether it is directly processed into small RNA species by Dicer, which cleaves long dsRNA molecules into short fragments as a first step in the RNAi pathway. Intriguingly, telomeric antisense transcripts, termed ?ARRET', have been detected in budding yeast (Houseley et al, 2007; Luke et al, 2008), although the canonical RNAi machinery does not exist in this organism.
Recent reports have also shown that TERRA may be regulated developmentally and, in turn, may be important for orchestrating some aspects of the complex chromosome transactions that occur during cellular differentiation (Schoeftner and Blasco, 2008; Marion et al, 2009; Zhang et al, 2009). Interestingly, in undifferentiated ES cells, TERRA can be found to be associated with both X chromosomes in females and with both X and Y chromosomes in males (Zhang et al, 2009). After differentiation, only one sex chromosome is marked with TERRA (Xi in females and Y in males,), indicating that TERRA localization is developmentally regulated. Consistently, when differentiated fibroblasts are induced to form pluripotent stem cells with the reprogramming factors, Oct3/4, Sox2, Klf4 and cMyc, TERRA levels are greatly increased (Marion et al, 2009). It is not yet clear whether the reprogramming itself is responsible for increased TERRA levels or whether the reprogramming factors are direct transcriptional regulators of TERRA.
Human cells depleted for UPF1, hEST1A and SMG1 increase the number of TERRA foci at chromosome ends and they lose entire telomeric tracts at a high rate. UPF1 depletion also causes severe DNA damage elsewhere in the genome and ATR-dependent checkpoint activation in the S phase (Azzalin and Lingner, 2006). The association of NMD factors with chromatin and their enrichment at telomeres suggest a causal link between proper TERRA displacement from telomeres and telomere integrity (Azzalin et al, 2007), and possibly the removal of other chromatin-associated RNA molecules by NMD factors elsewhere in the genome. Removal of TERRA and potentially other RNAs seems to be important for proper DNA replication and S-phase progression also in S. cerevisiae, as DNA replication defects were apparent in the rat1-1 mutant cells that upregulate TERRA (Luke et al, 2008). Replication might be impeded when TERRA hybridizes to single-stranded telomeric DNA, leading to the arrest of the replication fork and to the generation of double-stranded breaks in the telomeric tract (Figure 2). Interestingly, human UPF1 also physically interacts with DNA polymerase ? (Carastro et al, 2002; Azzalin and Lingner, 2006). Thus, one could speculate that DNA polymerase ? recruits UPF1 in conjunction with other SMG factors to displace TERRA from telomeres during progression of the replication fork. One speculative hypothesis would be that upon fork stalling and SMG recruitment to the replication fork, telomerase, through EST1A/SMG6, is also recruited in the event that de novo telomere addition is required after replication fork collapse to heal the broken telomere, or simply to ensure that telomerase is correctly deposited at the chromosome end in a timely manner (Figure 2). This model, as well as other models that may explain the observed phenotypes, needs to be tested in the future.
Several features seen in SMG-depleted cells were recently also detected in cells derived from ICF (Immunodeficiency, Centromeric region instability, Facial anomalies) patients, who carry defective genes for DNA methyltransferase 3b (DNMT3b). DNMT3b defects lead to hypomethylation of CpG dinucleotides in heterochromatic regions, including pericentromeric satellites, the inactive X chromosome and in subtelomeric regions. Intriguingly, ICF patient-derived cells also harbour short telomeres and display increased TERRA levels (Yehezkel et al, 2008). Furthermore, many chromosome ends in ICF patient-derived cells lacked detectable telomeric DNA signals altogether. Thus, as in the case of SMG depletion, TERRA upregulation correlates with a sudden telomere loss in ICF patient-derived cells.
The notion that the ends of our chromosomes are transcribed into TERRA and that telomeric chromatin contains RNA molecules adds another fascinating level of complexity and provides another example of a structural RNA molecule that may prove to be of utmost importance. TERRA and other chromatin-associated RNA molecules have the unique feature that they are synthesized at the site of their action. Thus, their synthesis rate is closely related to changes in the chromatin structure. Therefore, TERRA seems ideally suited to initiate an appropriate response at a telomere in reaction to other local changes at a telomere, which may involve length changes or perhaps cell-cycle-dependent changes. It has been proposed for the centromeric heterochromatin in fission yeast that a boost of transcription occurs in the S phase of the cell cycle in order to trigger assembly of the heterochromatin, which, when fully assembled, prevents further transcription at the centromere during other phases of the cell cycle (Kloc and Martienssen, 2008). Similarly, TERRA may trigger heterochromatin assembly at the telomere, contribute to the regulation of telomerase at individual chromosome ends in a length-dependent manner and, perhaps, even be involved in DNA damage response at short telomeres.
Several of the above-discussed biological functions of TERRA are still speculative, and the underlying mechanisms of action remain to be discovered. The telomeric dysfunctions of TERRA that can lead to sudden telomere loss are also very ill defined. Clearly, it is important to analyze the mechanisms of TERRA biogenesis and turnover, identify the protein factors that interact and collaborate with TERRA and elucidate their mechanisms of action. These studies may also uncover or show possible functions of TERRA during tumourigenesis or animal development. These are exciting times on planet earth (TERRA)?a feel of change is coming on for our views on how chromosome ends are regulated and how they function.
Work in JL's laboratory was supported by grants from the Swiss National Science Foundation, the Human Frontier Science Program, the EU 7th Framework Programme and by an ERC-advanced investigator grant.
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Keywords: chromatin, non-coding RNA, telomerase, telomeres, TERRA.
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