The heavily methylated state of subtelomeric regions, the gene-less nature of telomeres, and the observed telomere position effect led to the notion that telomeres are transcriptionally silent [5
]. This hypothesis was recently challenged when several groups independently demonstrated that subtelomeric and telomeric regions, although devoid of genes, have the potential to be transcribed into telomeric repeat-containing RNA (TERRA) [9
]. TERRA molecules have been identified in human, mouse, fish, and recently baker’s yeast, indicating that TERRAs are conserved among eukaryotes. TERRA transcripts are synthesized from the C-rich strand and polyadenylated, and their synthesis is α-amanitin-sensitive, suggesting that they are transcripts of RNA polymerase II (RNA Pol II) [9
]. Further support of a role for RNA Pol II in TERRA transcription derives from the observation that RNA Pol II is enriched at telomeres and associates with the telomeric DNA repeat-binding protein TRF1. TERRA transcripts can be found throughout the different stages of the cell cycle, and their levels are affected by several factors that include telomere length, tumor stage, cellular stress, developmental stage, and telomeric chromatin structure. TERRA most likely regulates telomere length directly since longer telomeres produce more TERRA molecules and thereby create a negative feedback by blocking telomere access to telomerase [10
]. However, it is largely unclear how the expression of TERRA and the amount of TERRA transcripts are regulated in the cell.
Insights into the possible regulation of TERRA synthesis came from experiments demonstrating that TERRA synthesis is upregulated by the mixed lineage leukemia (MLL) protein, which is the mammalian homologue of the Drosophila melanogaster
trithorax protein, and p53 after the induction of telomere uncapping [12
]. MLL is cleaved into two fragments by taspase 1: the N-terminal fragment associates the MLL complex to chromosomal sites, whereas the C-terminal fragment contains a SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain that manifests H3/K4 histone methytransferase (HMT) activity. Besides its established role as an HMT and general transcriptional regulator, MLL binds to telomeres. The binding of MLL to telomeres has been shown to affect telomere epigenetic status by methylating telomeric H3K4 [12
]. MLL also interacts with p53 to increase TERRA synthesis in response to progressive telomere uncapping [12
]. Therefore, MLL-dependent TERRA synthesis seems to represent a cellular response to telomere uncapping, thereby preventing DNA damage responses and the induction of cellular senescence.
Besides MLL, other proteins have been suggested to play a role in regulating TERRA levels either directly or indirectly [9
]. For instance, downregulating the cellular abundance of TRF1 has been shown to decrease the cellular abundance of TERRA without affecting the binding of RNA Pol II to telomeres [9
]. Furthermore, TERRA levels are increased in cells that are deficient for certain HMTs, whereas TERRA levels are decreased in cells lacking DNA methyl transferase (DNMT) proteins or Dicer [10
]. A role for these proteins in regulating TERRA levels suggests that the heterochromatic state of telomeric regions is important for the regulation of TERRA synthesis.
In addition, several factors (UPF1, EST1A/SMG6, and SMG1) of the mammalian-cell nonsense-mediated mRNA decay (NMD) machinery are enriched at telomeres and negatively regulate TERRA association with telomeric chromatin [9
], suggesting a link between TERRA, NMD factors, and telomere stability. Consistent with a role for these NMD factors in telomerase regulation, depletion of any one of these factors from cells not only increases TERRA binding to telomeres but also results in telomere damage and telomere loss [9
A first clue to how TERRA transcripts might regulate telomerase function came from experiments demonstrating that TERRA-mimetic oligonucleotides are able to inhibit human telomerase in vitro
]. This result suggests that TERRA might directly inhibit telomerase function by duplexing with the RNA moiety of the telomerase.
The finding that TERRA is also present in the genetically tractable Saccharomyces cerevisiae
was instrumental to further dissecting the processes that regulate TERRA synthesis and TERRA cellular abundance [11
]. Using yeast, Lingner and colleagues [11
] demonstrated that, in rat1-1
mutant cells, which are deficient in the Rat1p 5′-to-3′ exoribonuclease, much higher levels of TERRA could be detected. This result indicates that Rat1p, which functions in the decay of normal mRNAs and NMD targets, degrades TERRA RNAs [11
]. The association of Rat1p with telomeres suggests that Rat1p might directly degrade TERRA RNAs and provides the first evidence for a role of RNA degradation in the regulation of the TERRA transcriptome.
Like mammalian TERRAs, yeast TERRAs are transcribed by RNA Pol II since they (a) fail to accumulate in rat1-1
mutant cells in which RNA Pol II has been co-inactivated [11
] and (b) are polyadenylated by the poly(A) polymerase Pap1p [11
]. Since Pap1p adds poly(A) tails to pre-mRNAs during the process of mRNA biogenesis, a possible role during TERRA biogenesis for the same endonucleolytic cleavage factors that generate substrates for poly(A) addition during mRNA biogenesis was tested. Interestingly, TERRA levels were indeed decreased in rat1-1
cells harboring co-inactivated subunits of the endonucleolytic cleavage factor CF1A, demonstrating the importance of this factor in TERRA biogenesis [11
]. These results suggest that TERRA processing involves the Pap1p-mediated addition of poly(A) to TERRA 3′ ends that are generated by the same activities that generate pre-mRNA 3′ ends. Alternatively, the 5′ ends of TERRA that are generated by these activities could be degraded by the 5′-to-3′ exonucleolytic activity of Rat1p [11
]. Together, these results suggest an intimate interplay between mRNA-processing pathways and TERRA turnover. Further support for the regulation of telomerase function by Rat1p and TERRA comes from the observation that rat1-1
mutant cells have short telomeres. TERRA seems to inhibit telomerase activity directly since telomeres were shown to shorten at the same rate in telomerase-deficient cells that did or did not express Rat1p.
How could TERRA regulate telomerase activity? Two scenarios are conceivable. In one, TERRA could duplex with the RNA moiety of telomerase and directly inhibit telomerase activity. In the other, TERRA could associate with telomeric DNA repeats and block telomerase access to the telomeres. Support for the first mechanism comes from experiments described above demonstrating that TERRA-mimetic oligonucleotides inhibit telomerase activity [10
]. Support for the second scenario comes from the finding that overexpressing ribonuclease H in rat1-1
cells results in telomere lengthening. This observation suggests that telomerase function in rat1-1
cells is inhibited by RNA/DNA hybrids formed between TERRA and telomeric DNA [11
]. However, inhibition was less pronounced in cells with endogenous levels of ribonuclease H or in wild-type cells that overexpress ribonuclease H [11
]. In theory, both mechanisms could regulate telomerase activity at telomeric DNA ends, and future experiments will undoubtedly address existing uncertainties.
Together, these observations are of medical relevance because abnormal levels of TERRA and the resulting abnormal telomeric phenotypes are likely to affect processes such as hematopoesis, embryonic development, and stem cell biology. Indeed, increased TERRA levels have already been associated with human disease [14
]. As a case in point, mutations within the DNA methyltransferase 3b (DNMT3b) gene cause hypomethylation of satellite and non-satellite repeat regions, including subtelomeric regions, which leads to an autosomal-recessive disease called ICF (immunodeficiency, centromeric region instability, facial abnormalities) syndrome [15
]. In cells from ICF patients, hypomethylation of subtelomeric regions was recently associated with increased levels of TERRA synthesis and accelerated telomere shortening [14
]. These findings provide a first mechanistic explanation for the observed telomeric phenotype in ICF patients. However, these observations are in contrast to the finding that TERRA levels are decreased in cells deficient for DNMT1, DNMT3a, and DNMT3b [10
]. The use of different cell lines (mouse embryonic stem cells versus somatic cells from ICF patients) might account for the discrepancy.
Further support for TERRA RNA regulating telomerase activity comes from the observation that tumor-derived cell lines that rely on telomerase to maintain telomeres manifest a decreased level of TERRA RNA when compared with tumor-derived cells using the ALT (alternative lengthening of telomeres) mechanism [16
]. This decrease correlates mainly with increased cytosine DNA methylation at positions that reside adjacent to telomeres. Therefore, telomerase activity seemed to be tightly linked to subtelomeric DNA methylation, TERRA levels, and a telomeric heterochromatin state that represses telomeric transcription.