Telomeric Repeat-containing RNA Molecules Research Articles

Long non-coding RNA TERRA influences DNA damage and survival of endothelial cells and cardiomyocytes

Abstract Ageing is the major risk factor for cardiovascular disease. Current therapies are mainly based on proteins, while targeting long non-coding RNAs (lncRNAs) is largely unexplored. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. Although telomeres are heterochromatic regions, non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are transcribed from the telomeres of most chromosomes. The transcription of TERRA starts at the subtelomere and ends in the telomere, leading to molecules of 0.2–10kb. This study aims to characterize the role of TERRA in the cardiovascular system. TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). An increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donor heart tissue (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found in old passage human umbilical vein endothelial cells (HUVECs) (P15–17) compared to young HUVECs (P3) (p=0.014). IPSC-derived cardiomyocytes also increased the expression of h20q-TERRA with increasing passage (p=0.011). After knockdown of h20q-TERRA with LNA GapmeRs HUVECs show less sprout formation in a spheroid assay compared to negative control transfected HUVECs (p=0.002), without showing a change in migration (p=0.205) or proliferation (p=0.114). H20q-TERRA knockdown revealed that there was more apoptosis (p=0.015), more DNA damage as measured with the comet assay (p<0.001), increased γH2AX levels (p=0.029) and an increase in γH2AX colocalization with the telomere (p=0.011). The amount of phosphorylated P53 was also increased after knockdown (p=0.038), while it was decreased after TERRA overexpression (p=0.001). Inhibition of the ATM-yH2AX-P53 pathway did however not reduce the increased apoptosis after knockdown (KU60019 p=0.399; siP53 p=0.303). Silencing the m18-TERRA molecule in mouse endothelial H5V cells led to an increase in caspase activity (p=0.004) similar to what was shown in HUVECs. In addition, increased caspase activity (p=0.012), increased γH2AX levels (p<0.001) and increased γH2AX colocalization with the telomere (p=0.007) was also shown after silencing of h20q-TERRA in human cardiomyocytes. In summary, our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival. These data show that TERRA transcripts are induced in cardiovascular ageing and are essential for endothelial cell function. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): Horizon 2020

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

Genomic origin and nuclear localization of TERRA telomeric repeat-containing RNA: from Darkness to Dawn.

Long noncoding RNAs, produced from distinct regions of the chromosomes, are emerging as new key players in several important biological processes. The long noncoding RNAs add a new layer of complexity to cellular regulatory pathways, from transcription to cellular trafficking or chromatin remodeling. More than 25years ago, the discovery of a transcriptional activity at telomeres of protozoa ended the long-lasting belief that telomeres were transcriptionally silent. Since then, progressively accumulating evidences established that production of TElomeric Repeat-containing RNA (TERRA) was a general feature of eukaryotic cells. Whether TERRA molecules always originate from the telomeres or whether they can be transcribed from internal telomeric repeats as well is however still a matter of debate. Whether TERRA transcripts always localize to telomeres and play similar roles in all eukaryotic cells is also unclear. We review the studies on TERRA localization in the cell, its composition and some aspects of its transcriptional regulation to summarize the current knowledge and controversies about the genomic origin of TERRA, with a focus on human and mouse TERRA.

Telomeric Repeat Containing RNA (TERRA): Aging and Cancer

Telomeric repeat containing RNAs (TERRA) are small RNA molecules synthesized from telomeric regions which were previously considered as silent genomic domains. In normal cells, these RNAs are transcribed in a direction from subtelomeric region towards the chromosome ends, but in case of cancer cells, their expression remains limited or absent. Telomerase is a rate limiting enzyme for cellular senescence, cancer and aging. Most of the studies deal with the manipulation of telomerase enzyme in cancer and aging either by synthetic oligonucleotide or by natural phytochemicals. Here, we collected evidences and discussed intensely about the bio-molecular structure of TERRA, naturally occurring ligands of telomerase, and their genetic and epigenetic regulations in aging associated diseases. Due to their capability to act as naturally occurring ligands of telomerase, these RNAs can overcome the limitations possessed by synthetic oligonucleotides, which are aimed against telomerase. Drugs specifically targeting TERRA molecules could modulate telomerase-mediated telomere lengthening. Thus, targeting TERRA-mediated regulation of telomerase would be a promising therapeutic strategy against cancer and age-associated diseases.

Telomeric repeat-containing RNA TERRA: a noncoding RNA connecting telomere biology to genome integrity.

Telomeres are dynamic nucleoprotein structures that protect the ends of chromosomes from degradation and activation of DNA damage response. For this reason, telomeres are essential to genome integrity. Chromosome ends are enriched in heterochromatic marks and proper organization of telomeric chromatin is important to telomere stability. Despite their heterochromatic state, telomeres are transcribed giving rise to long noncoding RNAs (lncRNA) called TERRA (telomeric repeat-containing RNA). TERRA molecules play critical roles in telomere biology, including regulation of telomerase activity and heterochromatin formation at chromosome ends. Emerging evidence indicate that TERRA transcripts form DNA-RNA hybrids at chromosome ends which can promote homologous recombination among telomeres, delaying cellular senescence and sustaining genome instability. Intriguingly, TERRA RNA-telomeric DNA hybrids are involved in telomere length homeostasis of telomerase-negative cancer cells. Furthermore, TERRA transcripts play a role in the DNA damage response (DDR) triggered by dysfunctional telomeres. We discuss here recent developments on TERRA's role in telomere biology and genome integrity, and its implication in cancer.

Telomeric noncoding RNA: telomeric repeat‐containing RNA in telomere biology

Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and activation of DNA damage response. For this reason, functional telomeres are vital to genome stability. For years, telomeres were assumed to be transcriptionally silent, because of their heterochromatic state. It was only recently shown that, in several organisms, telomeres are transcribed, giving rise to a long noncoding RNA (lncRNA) called telomeric repeat-containing RNA (TERRA). Several lines of evidence now indicate that TERRA molecules play crucial roles in telomere homeostasis and telomere functions. Recent studies have shown that the expression and regulation of TERRA are dynamically controlled by each chromosome end. TERRA has been involved in the regulation of telomere length, telomerase activity, and heterochromatin formation at telomeres. The correct regulation of the telomeric transcripts may be essential to genome stability, and altered TERRA levels associate with tumorigenesis and cellular senescence. Thus, the study of the molecular mechanisms of TERRA biogenesis and function may advance the understanding of telomere-related diseases, including cancer and aging.

TERRA –A Calling Card for Telomerase

Transcription of telomeric DNA has long been thought to inhibit telomerase. Using live cell imaging, in this issue, Cusanelli et al. (2013) now reveal a different scenario: telomeric RNA is preferentially generated at short telomeres and delivers telomerase to the chromosome end from which the transcript originated.

A three-state model for the regulation of telomerase by TERRA and hnRNPA1

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5′-UUAGGG-3′ repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.

More on the Lack of Correlation between Terra Expression and Telomere Length

More on the Lack of Correlation between Terra Expression and Telomere Length

Telomeric repeat-containing RNA and telomerase in human fetal oocytes

What is the distribution of telomeric repeat-containing RNA (TERRA) and of telomerase in human fetal oocytes? TERRA forms discrete foci at telomeres of human fetal oocytes and it co-localizes with both the shelterin component telomeric repeat-binding factor 2 (TRF2) and the catalytic subunit of human telomerase at the telomeres of meiotic chromosomes. TERRA is a structural element of the telomeric chromatin that has been described in somatic cells of many different eukaryote species. The telomerase enzyme is inactive in adult somatic cells but is active in germ cells, stem cells and in the majority of tumors; however, its distribution in oocytes is still unknown. For this study, ovarian samples from four euploid fetuses of 22 gestational weeks were used. These samples were obtained with the consent of the parents and of the Ethics Committee of Hospital de la Vall d'Hebron. We analyzed the distribution of TERRA and telomerase in cells derived from human fetal ovaries. The co-localization of TERRA, telomerase and telomeres was performed by optimizing a combination of immunofluorescence (IF) and RNA-fluorescent in situ hybridization (RNA-FISH) techniques. The synaptonemal complex protein 3 (SYCP3), TRF2 and protein component of telomerase [telomerase reverse transcriptase (TERT)] were detected by IF, whereas TERRA was revealed by RNA-FISH using a (CCCTAA)(3) oligonucleotide. SYCP3 signals allowed us to identify oocytes that had entered meiosis and classify them into the different stages of prophase I, whereas TRF2 indicated the telomeric regions of chromosomes. We show for the first time the presence of TERRA and the intracellular distribution of telomerase in human fetal ovarian cells. TERRA is present, forming discrete foci, in 75% of the ovarian tissue cells and most of TERRA molecules (≈ 83%) are at telomeres (TRF2 co-localization). TERRA levels are higher in oocytes than in ovarian tissue cells (P = 0.00), and do not change along the progression of the prophase I stage (P = 0.37). TERRA is present on ≈ 23% of the telomeres in all cell types derived from human fetal ovaries. Moreover, ≈ 22% of TERRA foci co-localize with the protein component of telomerase (TERT). We present a descriptive/qualitative study of TERRA in human fetal ovarian tissue. Given the difficult access and manipulation of fetal samples, the number of fetal ovaries used in this study was limited. This is the first report on TERRA expression in oocytes from human fetal ovaries. The presence of TERRA at the telomeres of oocytes from the leptotene to pachytene stages and its co-localization with the telomerase protein component suggests that this RNA might participate in the maintenance of the telomere structure, at least through the processes that take place during the female meiotic prophase I. Since telomeres in oocytes have been mainly studied regarding the bouquet structure, our results introduce a new viewpoint of the telomeric structure during meiosis.

The telomeric transcriptome: From fission yeast to mammals

The ends of linear eukaryotic chromosomes are transcribed into different species of non-coding transcripts (the telomeric transcriptome), including TERRA (telomeric repeat-containing RNA) molecules; however, the functions associated with the telomeric transcriptome remain elusive. Experimental evidence accumulated during the past few years indicates that the transcriptional activity of telomeres is changed in cells in which the integrity of the telomeres or the heterochromatic state of chromosome ends is altered. On the contrary transcription of a telomere appears not to be influenced by its length. In this paper we briefly review the current state of knowledge on the composition, biogenesis, and regulation of the telomeric transcriptome from yeasts to humans. We also suggest a model in which TERRA is part of the DNA damage response triggered by dysfunctional telomeres and discuss the potential involvement of telomere transcription in the development of human pathologies.

The telomeric transcriptome of Schizosaccharomyces pombe

Eukaryotic telomeres are transcribed into telomeric repeat-containing RNA (TERRA). Telomeric transcription has been documented in mammals, birds, zebra fish, plants and budding yeast. Here we show that the chromosome ends of Schizosaccharomyces pombe produce distinct RNA species. As with budding yeast and mammals, S. pombe contains G-rich TERRA molecules and subtelomeric RNA species transcribed in the opposite direction of TERRA (ARRET). Moreover, fission yeast chromosome ends produce two novel RNA species: C-rich telomeric repeat-containing transcripts (ARIA) and subtelomeric transcripts complementary to ARRET (αARRET). RNA polymerase II (RNAPII) associates with pombe chromosome ends in vivo and the telomeric factor Rap1 negatively regulates this association, as well as the cellular accumulation of RNA emanating from chromosome ends. We also show that the RNAPII subunit Rpb7 and the non-canonical poly(A) polymerases Cid12 and Cid14 are involved in the regulation of TERRA, ARIA, ARRET and αARRET transcripts. We confirm the evolutionary conservation of telomere transcription, and reveal intriguing similarities and differences in the composition and regulation of telomeric transcripts among model organisms.

Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways

Telomeres are transcribed into telomeric repeat-containing RNA (TERRA), large, heterogeneous, noncoding transcripts which form part of the telomeric heterochromatin. Despite a large number of functions that have been ascribed to TERRA, little is known about its biogenesis. Here, we present the first comprehensive analysis of the molecular structure of TERRA. We identify biochemically distinct TERRA complexes, and we describe TERRA regulation during the cell cycle. Moreover, we demonstrate that TERRA 5' ends contain 7-methylguanosine cap structures and that the poly(A) tail, present on a fraction of TERRA transcripts, contributes to their stability. Poly(A)(-) TERRA, but not poly(A)(+) TERRA, is associated with chromatin, possibly reflecting distinct biological roles of TERRA ribonucleoprotein complexes. In support of this idea, poly(A)(-) and poly(A)(+) TERRA molecules end with distinct sequence registers. We also determine that the bulk of 3'-terminal UUAGGG repeats have an average length of 200 bases, indicating that the length heterogeneity of TERRA likely stems from its subtelomeric regions. Finally, we find that TERRA is regulated during the cell cycle, being lowest in late S phase and peaking in early G(1). Our analyses offer the basis for investigating multiple regulatory pathways that affect TERRA synthesis, processing, turnover, and function.

The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5′-UUAGGG-3′ repeats near their 3′-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5′-UUAGGG-3′ repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

CpG-island promoters drive transcription of human telomeres

The longstanding dogma that telomeres, the heterochromatic extremities of linear eukaryotic chromosomes, are transcriptionally silent was overturned by the discovery that DNA-dependent RNA polymerase II (RNAPII) transcribes telomeric DNA into telomeric repeat-containing RNA (TERRA). Here, we show that CpG dinucleotide-rich DNA islands, shared among multiple human chromosome ends, promote transcription of TERRA molecules. TERRA promoters sustain cellular expression of reporter genes, are located immediately upstream of TERRA transcription start sites, and are bound by active RNAPII in vivo. Finally, the identified promoter CpG dinucleotides are methylated in vivo, and cytosine methylation negatively regulates TERRA abundance. The existence of subtelomeric promoters, driving TERRA transcription from independent chromosome ends, supports the idea that TERRA exerts fundamental functions in the context of telomere biology.

The Rat1p 5′ to 3′ Exonuclease Degrades Telomeric Repeat-Containing RNA and Promotes Telomere Elongation in Saccharomyces cerevisiae

Vertebrate telomeres are transcribed into telomeric repeat-containing RNA (TERRA) that associates with telomeres and may be important for telomere function. Here, we demonstrate that telomeres are also transcribed in Saccharomyces cerevisiae by RNA polymerase II (RNAPII). Yeast TERRA is polyadenylated and stabilized by Pap1p and regulated by the 5' to 3' exonuclease, Rat1p. rat1-1 mutant cells accumulate TERRA and harbor short telomeres because of defects in telomerase-mediated telomere elongation. Overexpression of RNaseH overcomes telomere elongation defects in rat1-1 cells, indicating that RNA/DNA hybrids inhibit telomerase function at chromosome ends in these mutants. Thus, telomeric transcription combined with Rat1p-dependent TERRA degradation is important for regulating telomerase in yeast. Telomere transcription is conserved in different kingdoms of the eukaryotic domain.

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

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.