Noncoding RNAs controlling telomere homeostasis in senescence and aging

Abstract

Aging is a universal and time-dependent biological decline associated with a progressive deterioration of cells, tissues, and organs. Age-related decay can eventually lead to pathologies such as cardiovascular and neurodegenerative diseases, cancer, and diabetes. A prominent molecular process underlying aging is the progressive shortening of telomeres, the structures that protect the ends of chromosomes, culminating in cellular senescence. Noncoding (nc)RNAs are emerging as major regulators of telomere length homeostasis. In this review, we describe the impact of ncRNAs on telomere function and discuss their implications in senescence and age-related diseases. We discuss emerging therapeutic strategies targeting telomere-regulatory ncRNAs in aging pathologies.

Noncoding RNAs In Telomere Dynamics

Only ~2% of the mammalian genome is transcribed into protein-coding RNAs, and the remaining ~98% was long considered to be inactive material (“junk DNA”). However, with the arrival of new technologies and analytical tools, noncoding (nc)RNAs have emerged as rich, abundant, and potent regulators of gene expression programs in cellular processes like proliferation, apoptosis, differentiation, and senescence [1–3]. NcRNAs broadly include all RNAs from which there is no evidence of protein translation. The functions of regulatory RNAs become particularly relevant in times of cellular stress when transcription and translation are globally suppressed or restricted to certain mRNA subsets. In addition, they offer therapeutic opportunities that cannot be achieved by targeting coding RNAs (mRNAs).

The large and heterogeneous class of ncRNAs can be divided into two main groups according to their size: long ncRNAs, typically spanning several hundred nucleotides, and short RNAs, generally in the range of 20–30 nucleotides [4]. Among a broad range of mechanisms of gene regulation, some ncRNAs have been shown to regulate transcription by interacting with chromatin-modifying complexes or by interfering with or enhancing the transcriptional machinery [5–7]. Other ncRNAs modulate key post-transcriptional steps by influencing mRNA processing, transport, storage, degradation, and translation. Many ncRNAs have been annotated thus far, but our understanding of their roles in gene regulation is in its infancy.

Telomeres are protective structures at the end of linear chromosomes comprising repeats of the tandem sequence TTAGGG and the shelterin complex (TRF1, TRF2, POT1, RAP1, TIN2, TPP1). Telomeres prevent DNA damage and end-to-end fusions of chromosomes (Figure 1) and shorten progressively with each cell division due to a defect in the DNA replicative mechanism known as the ‘end-replication problem’. Upon removal of the last RNA primer at the 3′ end of the lagging strand, the newly synthesized strand will be a few nucleotides shorter, resulting in telomere shortening. The progressive loss of telomeres causes exposure of the DNA ends, eventually triggering a DNA Damage Response (DDR) that culminates with chromosomal instability and the formation of aberrant chromosome end-to-end fusions [8, 9].

Figure 1.

Schematic indicates the major proteins that protect telomeric integrity (Shelterin complex, green) and length (Telomerase complex, blue), as well as the lncRNAs GUARDIN, TERRA and TERC (blue font) and microRNAs (red font) that modulate telomere homeostasis by integrating the DNA Damage Response (DDR, grey), protecting telomere ends, and controlling telomerase activity.

Noncoding (nc)RNAs are emerging as major regulators of telomere length homeostasis. Here, we describe the impact of ncRNAs on the telomere biology and in particular their possible implications in senescence and age-related pathologies associated with telomere dysfunctions (Figure 1). Specifically, our discussion focuses on two types of regulatory RNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs).

Telomeres in Senescence and Age-related Diseases

The enzyme telomerase counteracts telomere shortening by replenishing telomeric repeats. In this manner, telomerase protects the genetic information from being truncated progressively during cell division [8–10]. However, most somatic cells have no detectable telomerase and undergo telomere shortening, which eventually leads to loss of telomere capping and triggers DDR. In cells with functional checkpoints, the DDR leads to increased levels of the transcription factor TP53 (p53), which is involved in DNA repair, cell cycle arrest, and apoptosis. TP53 transcriptionally induces the production of p21 (CDKN1A), a cyclin-dependent kinase (CDK) inhibitor that can halt cell division [9, 10]. Additionally, telomere dysfunction may also increase the levels of the CDK inhibitor p16 (CDKN2A) which further suppresses cell growth. Both p21 and p16 enable the function of the retinoblastoma protein (RB) and reinforce the cell cycle arrest [11, 12]. Therefore, in cells with functional cell cycle regulators, critical telomere shortening is associated with reduced proliferative capacity. Cells then enter a state of indefinite growth arrest and become senescent, although they remain metabolically active.

By contrast, impairment of checkpoints and tumor suppressor pathways causes cells to continue dividing and escape from replicative senescence. Fully unprotected chromosome ends can form end-to-end fusions leading to genomic instability and a state of “crisis” associated with massive cell death. Given that the reorganization of the genome allows the generation of pre-neoplastic cells [8–10], most tumor cells have devised mechanisms to maintain telomeres of a certain minimal size by reactivating the telomerase reverse transcriptase, thus expanding their replicative potential. The enzyme telomerase is composed of two core components, the telomerase reverse transcriptase (TERT) and the telomerase RNA (TERC, TElomerase RNA Component, also known as hTR), which serves as the template for TERT in the addition of telomeric repeats. The active telomerase complex requires other ancillary components, including the binding partners dyskerin (DKC1), NOP10, NHP2, GAR1, and Telomerase Cajal body protein 1 (TCAB1) [13]. In a small percentage of telomerase-negative immortalized cells and cancer cells, alternative lengthening of telomere (ALT) is achieved through homologous recombination-dependent synthesis of new telomeric DNA [9].

Senescent cells have been detected in multiple age-related diseases and are associated with loss of tissue function during the aging process. Besides cell cycle arrest, senescent cells undergo changes in morphology, gene expression patterns, and metabolic programs. Importantly, senescent cells display a distinct trait known as the senescence-associated secretory phenotype (SASP), whereby they secrete high levels of pro-inflammatory cytokines capable of altering the microenvironment, triggering immune surveillance, enhancing angiogenesis, and remodeling the extracellular matrix, with a range of beneficial and deleterious effects on different organs [14–17]. The causes, consequences, and phenotypes of cell senescence have been reviewed in detail [17–20].

Cellular senescence may also occur independently of telomere shortening; for example, DNA damage, oxidative injury, and oncogenic signaling are able to induce cellular senescence. Stress- and oncogene-induced senescence may also incur acute damage at the telomeres and trigger DDR signaling, in turn suppressing cell proliferation [21–23]. According to this evidence, telomeres not only determine the rounds of division of a cell, but also act as sensors of intrinsic and extrinsic stresses to suppress the proliferation of cells that have accumulated significant genomic damage [10].

Several lines of evidence support the notion that short telomeres are associated with premature aging and age-related diseases [10, 24]. First, an inverse association exists between chronological age and telomere length, even though telomere length is highly heterogeneous among different tissues and different individuals of the same age [24, 25]. We note that it is unclear whether telomere length is a useful biomarker of aging and cellular senescence, as discussed elsewhere [24–28]. Second, accelerated aging conditions known as Short Telomere Syndromes (STSs) are caused by gene mutations resulting in premature telomere attrition and multisystemic diseases such as Dyskeratosis Congenita, Werner’s Syndrome, Hutchinson-Gilford Syndrome and Ataxia-telangiectasia. Patients with these syndromes often show phenotypes of accelerated aging such as diabetes, myocardial infarction, and cognitive decline [29, 30]. Third, telomere shortening has been associated with common age-related diseases including cardiovascular disease, diabetes, neurodegeneration, chronic obstructive pulmonary disease (COPD), and skin disorders [25, 31]. Fourth, many age-related diseases are exacerbated by immune cell senescence. For these reasons, the study of telomere attrition in immune cells like leukocytes is relevant for the etiology of these conditions and may serve as surrogate of telomere length in other tissues [29]. Alterations in leukocyte telomere length (LTL) is associated with increased risk of many cancer types [25, 29, 31, 32], supporting the notion that premalignant cells have lost telomere length homeostasis, prior to the activation of telomerase-mediated telomere maintenance. In sum, understanding the molecular processes underlying telomere dysfunction could help identify possible therapeutic targets in age-related diseases.

microRNAs Regulating Telomere Dynamics in Senescence and Aging

MicroRNAs (miRNAs) are a major class of short ncRNAs that regulate gene expression programs post-transcriptionally. They generally function by binding to specific mRNAs through complementary sequences and inducing degradation and/or blocking the translation of the target mRNA [33]. Many microRNAs control the expression of factors that influence telomere length dynamics (Figure 1). In this section, we focus on the senescence-associated microRNAs (SA-miRNAs) that have been shown to play a role in senescence or aging by influencing telomere maintenance (Table 1).

Table 1.

Main microRNAs identified as regulators of telomere metabolism with a role in senescence and aging.

microRNA	Target mRNAs and pathways	Levels in senescence or aging	Ref.
miR-23a	TRF2	↑	[34, 35]
miR-34a	PNUTS FoxM1/MYC/TERT pathway SIRT1, SIRT6 SIRT7	↑	[38–43]
miR-195	TERT, SIRT1	↑	[44, 45]
miR- 126	Inhibited telomere-TP53-p21-RB and JAK/STAT pathways VCAN (versican), proteoglycan involved in the aging process	↓	[46, 47]
miR-155	TRF1	↓	[48, 49]
miR-22, miR-138, miR-181a, miR-217, miR449, miR-570	SIRT1	↑	[39, 50–55]
miR-26a, miR-145a	SIRT6 and SIRT2 in dysfunctional telomeres	↑	[40]
miR-766	SIRT6	↑	[56]
miR-128	TERT	↑	[57]
miR-296–5p, miR-512–5p	TERT	↑	[58, 59]
miR-150	DKC1	↑	[60]
miR-143, miR-145, miR-146	Can be regulated by expression of ectopic telomerase	↑	[49]
miR-let-7d-5p, miR-let-7e-5p, miR-23a-3p, miR-34a-5p, miR-125a-3p, miR-125a-5p, miR-125b-5p, miR-181a-5p	Increased in senescent fibroblasts, these microRNAs reduced production of cell cycle regulatory factors	↑	[61]

miR‐23a.

MicroRNA miR-23a was initially found to promote cellular senescence in human fibroblasts by inhibiting the expression of TRF2, a key component of the shelterin complex (Figure 1) [34]. In a later study, Satoh et al. [35] showed that high levels of miR-23a not only induced telomere shortening, but were also associated with poor clinical outcomes in patients with coronary artery disease (CAD), an age-related condition. Furthermore, the high levels of miR-23a in the CAD group were inversely correlated to the levels of TRF2 protein [35].

miR-34a.

This microRNA was recognized as a major regulator of the levels of surtuins, a family of NAD-dependent deacetylases that perform a variety of functions in several cellular compartments, including histone modification and control of metabolic proteins. Among them, SIRT1, SIRT6 and SIRT7, may localize at chromosome ends and maintain telomere length. Previous studies have shown that the deacetylase activity of sirtuins promotes telomere integrity by maintaining chromatin condensation and by promoting the interaction between histones and telomere regulatory factors, like components of the DNA repair machinery. Moreover, sirtuins suppress the DNA damage response at the telomere by inactivating TP53 [36, 37]. MiR-34 was recognized as a major repressor of the levels of SIRT1, SIRT6 and SIRT7 [38–40].

In cardiomyocytes, miR-34a modulated telomere maintenance by reducing the abundance of protein Phosphatase 1 NUclear Targeting Subunit (PNUTS, also known as PPP1R10), which directs protein phosphatase 1 (PP1) to the nucleus. PNUTS was previously found to inhibit the DDR and to interact with TRF2, further preventing telomere shortening [41, 42]. Furthermore, miR-34a induced senescence of human hepatocellular carcinoma (HCC) cells by modulating telomerase activity. In HCC tumors, the levels of miR-34a correlated inversely with telomere length and telomerase activity, while increasing miR-34a ectopically in HCC cells triggered senescence and affected cell viability. The actions by miR-34a on telomere maintenance were attributed to the miR-34a-mediated suppression of the Forkhead Box M1 (FOXM1) and MYC pathway, required for hTERT transcription and telomerase activity [43].

miR-195.

Compared to young mesenchymal stems cells (YMSCs), in which telomerase is highly abundant and active, old mesenchymal stem cells (OMSCs) express high levels of miR-195. The 3’UTR of TERT mRNA was identified as a putative target of miR-195 and depletion of miR-195 rescued hTERT expression and cell proliferation in OMSCs. Importantly, injection of OMSCs with depleted miR-195 significantly improved the regeneration of mouse heart after infarction [44]. A further study showed that miR-195 promoted senescence in skeletal muscle cells by lowering the production of SIRT1 [45]

miR-126.

A rise in miR-126 abundance delayed the senescence of human glomerular mesangial cells (HGMCs) induced by high glucose. While high glucose led to shortened telomeres, miR-126 upregulation was associated with extended telomeres and decreased expression of DNA damage response and /or senescence markers TP53 and p21 [46]. Another study found that deletion of miR-126a promoted hepatic aging in mice and induced age-associated telomere shortening [47].

miR-155.

MiR-155 was found elevated in breast cancer specimens, together with reduced levels of TRF1, a protein component of the shelterin complex (Figure 1). Accordingly, in breast cancer cells it was shown that miR-155 interacts with the 3’UTR of the TRF1 mRNA and represses the translation of the TRF1 protein. The increased levels of miR-155 were associated with telomere and genomic instability, and with poor clinical outcome in estrogen receptor-positive breast cancer [48]. Notably, miR-155 levels are often reduced in senescent cells [49].

Other microRNAs.

Additional microRNAs (miR-570, miR-217, miR-138, miR-22, miR-181a, miR-449, mir-26, mir-145a, miR-766) were reported to suppress the expression of sirtuin family members SIRT1 and SIRT6 [39, 40, 50–56], while other microRNAs modulate the expression of telomere components TERT (miR-128, miR-296–5p, and miR-512–5p) [57–59] and DKC1 (miR-150) [60]. Interestingly, some microRNAs were themselves regulated by hTERT levels in senescent cells, although the mechanisms are not well understood (miR-143, miR-145, miR-146 [49]). Finally, the expression levels of a subset of microRNAs (let-7d-5p, let-7e-5p, miR-125a-3p, miR-125a-5p, miR-125b-5p, miR-23a-3p, miR-34a-5p, miR-181a-5p) increased upon shortening of telomeres in senescent fibroblasts, and they in turn suppressed the production of various cell cycle regulatory transcripts [61].

Taken together, microRNAs can promote or suppress telomere function by modulating the response of telomeres to DNA damage, by suppressing the levels of proteins that protect telomere ends, and by influencing the levels of telomerase components (Figure 1).

lncRNAs Impacting Telomere Maintenance in Senescence and Aging

Several lncRNAs have been associated with the integration of the DDR, the protection of telomere ends and the maintenance of telomere length (Figure 1).

TERC (hTR)

The telomerase core component hTR (human telomerase RNA) is encoded by the TERC gene, located on the chromosomal region 3q26. TERC serves as a template for TERT by providing a sequence (AAUCCC) for the insertion of the repetitive G-rich DNA sequence 5′-TTAGGG-3′ to the ends of chromosomes [62]. TERC is involved in the localization and assembly of the telomerase holoenzyme, and modifications of specific TERC residues were shown to compromise the catalytic activity of the telomerase [63]. TERC contains three major structural and functional domains: a core domain featuring the template sequence, a Stem Terminus Element (STE, featuring conserved regions CR4/CR5 domain) essential for the telomerase catalytic activity, and a 3’ terminal domain (known as H/ACA small Cajal body-specific RNA) that contains specific signals for TERC processing and localization [63]. In 1997, the development of a telomerase-deficient (TERC^−/−) mouse provided a model for the study of telomere function in cancer, cardiovascular disease, and other age-related pathologies. The phenotype of late generations of TERC ^−/− mice included chromosome end fusions, decreased lifespan, and typical features of aging like atrophy and reduced angiogenesis [64, 65].

Genetic variations of the human TERC gene, as well as other telomere maintenance genes, can alter the stability and catalytic activity of the telomerase complex. In line with this notion, TERC mutations have been linked to telomere biology disorders [66] and to shorter human lifespan [67]. For example, mutations in TERC cause autosomal forms of dyskeratosis congenita (DC), characterized by shorter telomeres, cancer disposition, bone marrow failure, and premature aging. Although disease-associated TERC mutations are distributed throughout the RNA, the majority of them map to sequences essential for telomerase catalysis, a specific TERT-binding site, and the template region, causing reduced catalytic activity and aberrant addition of telomeric repeats [68].

Shorter telomeres were associated with the pathogenesis and development of age-associated cognitive decline and Alzheimer’s disease (AD). In particular, TERC genetic variations in AD patients were compared with the frequencies observed in control patients. Specific combinations of single-nucleotide polymorphisms (SNPs) of the TERC gene were found to affect the age at AD onset [69].

In addition, specific TERC mutations were found to be associated with a higher risk of Idiopathic Pulmonary Fibrosis (IPF), an aging-related syndrome characterized by interstitial lung scarring, as well as premature hair greying, bone marrow failure, and liver cirrhosis. IPF is thought to arise from the impaired regeneration of damaged alveolar epithelial cells in the presence of diminished telomerase function [70–73].

A recent study identified an alternative regulatory role for TERC on senescence and aging independent of its function in the telomerase complex [74]. TERC was found to be imported into mitochondria and processed to a shorter form, TERC-53, that was exported back to the cytosol. TERC-53 accumulated in the cytosol upon cellular stress and may serve as an indicator of mitochondrial function [74, 75]. Interestingly, overexpression of TERC-53 in human fibroblasts induced cellular senescence, and overexpression of mTerc-53 in mice accelerated cognitive decline; these interventions did not affect telomerase activity, but rather caused stem cell exhaustion and proliferative decline [74].

Recent evidence indicates that the mechanisms adopted by tumor cells to activate telomerase and avoid telomere shortening include amplification of the TERC gene, besides the well-known derepression of the TERT gene. In this manner, telomerase activation contributes to the evasion of cancer cells beyond the normal limits of proliferation [76, 77].

TERRA

Despite being heterochromatic, telomeric regions are transcribed into lncRNAs collectively known as TERRA (TElomeric Repeat-containing RNA) that play crucial roles in telomere protection and maintenance. TERRA transcription starts from the subtelomeric region and then proceeds into telomere repeats. In mammals, TERRA molecules have different lengths, ranging between 100 nt and 9 kb [78, 79].

TERRA transcripts can protect chromosome ends by promoting telomere maintenance [80, 81]. The G-rich 3′ end of TERRA folds into multimeric G-quadruplex structures composed of four G rings that interact with several capping molecules located at the telomere ends, such as the shelterin components TRF1 and TRF2 [82–85]. TERRA RNAs also interact with proteins that contribute to maintaining the heterochromatic state, including the Polycomb Repressive Complex 2 (PRC2), which has histone methyltransferase activity [86, 87]. The interaction between TERRA transcripts and PRC2 enhances the deposition of heterochromatin marks like H3K9me3, H3K27me3, H4K20me3 and HP1 at chromosome ends [86, 87]. At extratelomeric sites, the interaction between TERRA and epigenetic factors can regulate chromatin remodeling and transcription [88]. Finally, TERRA transcripts form RNA-DNA hybrid structures named R-loops, which influence telomere heterochromatin assembly, replication, and homologous recombination among telomeres, and can thereby impact the onset of senescence [88–91].

TERRA RNAs have been shown to modulate the activity of the telomerase. As TERRA transcripts contain tandem G-rich sequences (UUAGGG) with strong affinity for TERC [92], they may act as direct inhibitors of telomerase activity [93]. Moreover, TERRA transcripts may bind TERT directly and modulate telomerase activity independently of TERC [93]; accordingly, TERRA-mimicking oligonucleotides inhibited telomerase activity [94], while TERRA depletion increased it [88]. However, the role of TERRA on telomerase activity remains controversial, with evidence that TERRA transcripts were capable of recruiting telomerase to critically short telomeres to promote telomere elongation [95], and that high levels of TERRA did not affect telomere elongation in human cancer cells [96].

The molecular functions of TERRA on telomere maintenance in senescence are also unclear. On one hand TERRA may protect telomere ends, but on the other it can counteract telomere elongation. Overexpression of TERRA in telomerase‐negative cells delayed the onset of senescence [91], and masked 3’ overhangs of uncapped telomeres during DNA damage‐induced senescence, thus protecting telomere ends [97]; however, in other studies high levels of TERRA triggered premature senescence by suppressing telomere elongation and DNA replication [98, 99].

The role of TERRA in aging is also poorly understood. A recent review [100] suggested that an interplay between TERRA and TERC regulates telomerase activity and the survival rate of neural stem cells during aging. Therefore, shifts in abundance or activity of these molecules might be involved in age-associated changes [100]. Other studies revealed that TERRA levels were high in blood mononuclear cells of patients with IPF, while TERRA silencing improved telomere function [70, 101].

The levels of TERRA transcripts vary widely and differ among stages of cancer progression [102]. TERRA transcripts were reduced in telomerase-positive cancer cells, where the subtelomeric translational regions are highly methylated [103], but they were elevated in ALT-positive cancers [95, 102, 103]. These differences may be related to the fact that telomeric chromatin is less compact in ALT-positive cells than in telomerase-positive cells [80, 103].

In summary, even though TERRA functions have not yet been fully elucidated, the tight regulation of TERRA levels production appears to be necessary for maintaining telomere homeostasis, with consequences on cancer, senescence, and aging.

GUARDIN

The lncRNA GUARDIN, a transcriptional target of TP53, was shown to be important for maintaining genomic integrity both in unstimulated cells and in cells responding to DNA damage. GUARDIN protected telomere ends from damage and prevented chromosome end-to-end fusion in large part by sequestering miR-23a, and thereby ensuring the production of the shelterin component TRF2. In addition, GUARDIN promoted DNA repair by acting as a scaffold to facilitate the heterodimerization of breast cancer type 1 susceptibility protein (BRCA1) and the BRCA1-associated RING domain 1 (BARD1) protein, which stabilizes BRCA1. GUARDIN silencing not only induced apoptosis and cellular senescence, but it also reduced the growth of cancer xenografts in mice and sensitized cancer cells to genotoxic drugs [104].

Therapies Directed at Telomere-Regulatory ncRNAs: Progress and Prospects

The past decade has seen an escalation in the development of therapeutic approaches exploiting noncoding RNAs, mainly microRNAs and lncRNAs [105, 106]. Some interventions directed at microRNAs may be advantageous because a microRNA may jointly suppress multiple proteins implicated in a given phenotype, or it may suppress proteins selectively expressed in a specific cell type. Current therapeutic strategies to overexpress a specific miRNA involves the delivery of synthetic oligoribonucleotides that mimic the native miRNA, often bearing modifications and complexed molecules for stability and efficacy. Conversely, suppression of microRNA actions often relies on the delivery of antisense oligonucleotides that neutralize the endogenous microRNA and block its activity [107, 108].

Similarly, lncRNAs represent a class of attractive drug targets, given their dysregulation in disease and their tissue-specific expression. For example, many lncRNAs interact with the repressive PRC2 machinery and thus may have a broad impact on gene expression patterns. In this regard, different oligonucleotides have been developed to induce cleavage of a specific lncRNA or to compete with PRC2 for the association with a target lncRNA [106, 109].

Major challenges in RNA-directed oligonucleotide therapeutics are the toxicity at the therapeutic dose, the binding to unintended targets, the stability of the oligonucleotide, and the delivery to the intended tissue. Nanotechnology-based delivery systems have improved greatly the stability and target tissue specificity of therapeutic RNAs, while chemical modifications have improved the pharmacokinetic properties of the oligonucleotides and have lessened their toxicity and off-target actions [105, 106].

Several RNA-based therapeutics have been developed to treat age-related pathologies like cardiovascular disease, diabetes, neurodegeneration, and cancer [105]. Interestingly, some of the ncRNAs targeted therapeutically influence telomere maintenance. For example, in preclinical studies, the miR-34a mimic MRX34 reduced the growth of non-small cell lung cancer in mice and prolonged survival [110]. However, the phase 1 clinical trial of MRX34 was terminated due to adverse immune complications in some patients (NCT01829971)^I . Anti-miR-155 molecules are being investigated for the treatment of cutaneous T cell lymphoma (CTCL) and have successfully passed through phase 1 of clinical trials (NCT03837457) ^II . MiR-155 inhibitors are currently investigated also for the treatment of neuroinflammatory and neurodegenerative diseases [111].

LncRNAs linked to telomere function are also being evaluated as therapeutic targets. Given earlier evidence that inhibiting telomerase could be an effective therapeutic approach in various cancers, oligonucleotides or small molecules specifically directed at TERRA and TERC are being developed with the hope of modulating telomerase activity and telomere length. For example, the oligonucleotide Imetelstat binds with high affinity the template region of TERC, directly inhibiting telomerase activity. Phase 1 trials were successfully completed and clinical phase 2 trials are underway (NCT02598661) ^III . Strategies directed at TERC inhibition include the design of hammerhead ribozymes, RNA molecules able to cleave the target RNA in a site-specific manner, resulting in a reduced telomerase activity [112, 113].

TERRA RNAs may negatively regulate the telomerase activity and are often dysregulated in cancer and age-related diseases. Sinha et al. [102] recently reviewed several small molecules, like BRACO-19, which possess anti-cancer potential and are able to stabilize the telomeric DNA G-quadruplexes [114]. Given that ligand-induced telomeric G-quadruplex stabilization was shown to inhibit the telomerase activity, it will be interesting to investigate whether TERRA G-quadruplexes might be involved in the proposed mechanism and could be therapeutic targets.

In closing, the development of oligonucleotides, small molecules and other strategies targeting ncRNAs that modulate telomere function, together with the advancements in delivery systems, will further help in the development of new treatments against cancer and other age-related diseases (see Clinician’s Corner).

CLINICIAN’S CORNER.

• An important feature of aging is the progressive accumulation of senescent cells in some tissues, with negative consequences on organ homeostasis and function.

• Senescent cells are characterized by many different hallmarks including a state of indefinite cell cycle arrest and dysregulation of telomere maintenance. Cancer cells are able to overcome replicative senescence by reactivating the telomerase enzyme, thus acquiring aberrant cell proliferation.

• The molecular processes underlying age-associated diseases involve a multitude of noncoding RNAs with crucial roles in cellular processes. New interventions directed at enhancing or suppressing noncoding RNAs are being developed, some of which have already entered human clinical trials. There is mounting interest in RNA-centered strategies to intervene in age-related pathologies such as cancer and neurodegeneration.

• Therapeutic approaches targeting ncRNAs include RNA interference, antisense oligonucleotides and small molecules. Chemical modifications and innovative delivery systems are being developed to improve the stability and effectiveness of these molecules.

Concluding Remarks

NcRNAs are major regulators of telomere dynamics, which are well-known for playing an essential role in cellular senescence, aging and cancer. We have described the broad, pleiotropic action of miRNAs in several pathways involved in telomere homeostasis. We have also reported the role of specific lncRNAs in chromosome ends protection and regulation of the telomerase activity. However, despite progresses made in RNA studies, several questions still need to be addressed (see Outstanding Questions). Besides chromosome ends protection and DNA damage sensing, telomeres play an important role in other biological processes. An area of telomere research which has been unexplored for long time is the chromosomal looping between telomeres and distant genic regions. This long-distance interaction was shown to regulate gene expression and was shown to modulate, among other transcripts, the expression of ncRNAs [115]. Whether ncRNAs might in turn regulate the process of telomere looping in long-distance interactions remains to be determined.

OUTSTANDING QUESTIONS BOX.

What other cellular processes are regulated by telomeres?

Telomeres are more than just a clock, shortening after each cell division. They protect the genetic information from being truncated during DNA replication, prevent the fusion of chromosomes, and suppress the proliferation of cells with genomic damage. Telomeres may also regulate gene expression through long-distance interaction with other DNA regions. Whether or not ncRNAs are implicated in these trans-acting functions of telomeres awaits investigation.

Are there ubiquitous and tissue-restricted telomeric ncRNAs?

We have limited knowledge of the molecular mechanisms by which ncRNAs regulate telomere homeostasis in different cell types. A comprehensive understanding of the molecules and pathways whereby ncRNAs control telomere homeostasis constitutively and in specific tissues will enable more precise interventions.

What disease processes are amenable to therapies targeting telomere-related ncRNAs?

Given that senescent cells have been implicated in many declines and pathologies of aging, interventions aimed at stabilizing or exposing telomeres could prove to be valuable therapeutic tools. With an expanding number of ncRNAs successfully targeted in disease conditions, telomeric ncRNAs are becoming attractive therapeutic targets when senescent cells contribute to disease pathogenesis.

Most studies have focused on the role of telomeres in tumor suppressor mechanisms and cellular senescence. For this reason, an area of particular interest is the development of therapeutic strategies targeting telomeric ncRNAs in cancer and age-related diseases. Notably, some of these compounds have already passed the first phase of clinical trials. Future research will probably identify tissue-specific ncRNAs with a role in telomere dynamics, allowing a more precise intervention in the context of therapeutic strategies.

HIGHLIGHTS.

• Senescent cells accumulating in aging tissues display indefinite cell cycle arrest, shortened telomeres, and enhanced secretion of pro-inflammatory factors.

• Non-coding RNAs critically regulate gene expression programs in many physiological and pathological processes, including those associated with aging.

• Non-coding RNAs regulate telomere homeostasis, either directly or indirectly, and thereby coordinate molecular processes inherent to cellular senescence and aging.