Telomeric Repeat-containing RNA Expression Research Articles

The RNA-binding motif protein 14 regulates telomere integrity at the interface of TERRA and telomeric R-loops.

Telomeric repeat-containing RNA (TERRA) and its formation of RNA:DNA hybrids (or TERRA R-loops), influence telomere maintenance, particularly in human cancer cells that use homologous recombination-mediated alternative lengthening of telomeres. Here, we report that the RNA-binding motif protein 14 (RBM14) is associated with telomeres in human cancer cells. RBM14 negatively regulates TERRA expression. It also binds to TERRA and inhibits it from forming TERRA R-loops at telomeres. RBM14 depletion has several effects, including elevated TERRA levels, telomeric R-loops, telomere dysfunction-induced DNA damage foci formation, particularly in the presence of DNA replication stress, pRPA32 accumulation at telomeresand telomere signal-free ends. Thus, RBM14 protects telomere integrity via modulating TERRA levels and its R-loop formation at telomeres.

RBMX involves in telomere stability maintenance by regulating TERRA expression.

Telomeric repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from subtelomeric to telomeric region of chromosome ends. TERRA is prone to form R-loop structures at telomeres by invading into telomeric DNA. Excessive telomere R-loops result in telomere instability, so the TERRA level needs to be delicately modulated. However, the molecular mechanisms and factors controlling TERRA level are still largely unknown. In this study, we report that the RNA binding protein RBMX is a novel regulator of TERRA level and telomere integrity. The expression level of TERRA is significantly elevated in RBMX depleted cells, leading to enhanced telomere R-loop formation, replication stress, and telomere instability. We also found that RBMX binds to TERRA and the nuclear exosome targeting protein ZCCHC8 simultaneously, and that TERRA degradation slows down upon RBMX depletion, implying that RBMX promotes TERRA degradation by regulating its transportation to the nuclear exosome, which decays nuclear RNAs. Altogether, these findings uncover a new role of RBMX in TERRA expression regulation and telomere integrity maintenance, and raising RBMX as a potential target of cancer therapy.

Cohesin SMC1β promotes closed chromatin and controls TERRA expression at spermatocyte telomeres.

Previous data showed that meiotic cohesin SMC1β protects spermatocyte telomeres from damage. The underlying reason, however, remained unknown as the expressions of telomerase and shelterin components were normal in Smc1β -/- spermatocytes. Here. we report that SMC1β restricts expression of the long noncoding RNA TERRA (telomeric repeat containing RNA) in spermatocytes. In somatic cell lines increased TERRA was reported to cause telomere damage through altering telomere chromatin structure. In Smc1β -/- spermatocytes, we observed strongly increased levels of TERRA which accumulate on damaged chromosomal ends, where enhanced R-loop formation was found. This suggested a more open chromatin configuration near telomeres in Smc1β -/- spermatocytes, which was confirmed by ATAC-seq. Telomere-distal regions were not affected by the absence of SMC1β but RNA-seq revealed increased transcriptional activity in telomere-proximal regions. Thus, SMC1β promotes closed chromatin specifically near telomeres and limits TERRA expression in spermatocytes.

Long non-coding RNA TERRA regulates DNA damage and survival of endothelial cells and cardiomyocytes: implications for aging

Abstract Introduction Ageing is the major risk factor for cardiovascular disease. Long non-coding RNAs are emerging as novel regulators of cellular functions and contributors to cardiovascular ageing. One of the hallmarks of aging is telomere attrition. Non-coding transcripts called Telomeric repeat-containing RNA (TERRA) are molecules of 0.2–10kb in length which are transcribed from the subtelomeres and telomeres of chromosomes and might play a role in cardiovascular ageing. Purpose This study aims to characterize the role of TERRA in aging of the cardiovascular system. Methods and results TERRA molecules from different chromosomes were upregulated in the hearts of old mice compared to young mice (p=0.002). Increased TERRA expression was also shown in heart tissue of patients with ischemic heart disease compared to donors (p=0.001). In vitro an upregulation of the TERRA molecule transcribed from chromosome 20 (h20q-TERRA) was found with increasing passage in human umbilical vein endothelial cells (HUVECs) (p=0.014) and IPSC-derived cardiomyocytes (p=0.011). After h20q-TERRA knockdown 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 an increase in apoptosis (p=0.015) and telomeric DNA damage (p=0.011) and a decrease in telomere length (p<0.001), while lentiviral TERRA-repeat overexpression had the opposite effect (p=0.016, p=0.031, p<0.001, resp.). Apoptosis (p=0.012) and telomeric DNA damage (p=0.007) were also increased after the knockdown of h20q-TERRA in human cardiomyocytes. An apoptosis pathway profiler array in HUVECs showed that the expression of the antioxidant PON2 was decreased after knockdown of h20q-TERRA (p=0.040). PON2 expression was increased after TERRA overexpression (p=0.003). RNA immunoprecipitation revealed that TERRA can bind to PON2. Silencing PON2 in TERRA overexpressing cells diminished the TERRA-mediated decrease in caspase activation, suggesting a detrimental role for PON2 in caspase activation and endothelial cell survival. Conclusion Our data demonstrates that TERRA is upregulated with ageing and plays a role in endothelial and cardiomyocyte function and survival. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Horizon 2020

TERRA R-LOOPS ARE INCREASED IN ACUTE MYELOID LEUKEMIA

Telomeric repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from subtelomeric regions towards telomere ends. TERRA binds to subtelomeric and telomeric regions, forming R-loops, structures composed of DNA-RNA hybrids and displaced single-stranded DNA. TERRA R-loops protect telomeres from DNA damage and promote telomere elongation in short telomeres by enabling telomerase or by activating alternative telomere lengthening. However, high or low TERRA levels are associated with DNA damage and telomere dysfunction. RNaseH1 and RNaseH2 cleave R-loops at telomeres, and regulate R-loops levels. The role of TERRA R-loops in telomere maintenance in human cells is not well understood, since conflicting results derive from experiments using diverse organisms and commercial cell lines, with few studies applying primary human cells. Here we describe for the first time the TERRA physiology in hematopoietic cells of patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), telomeropathies (TEL), and healthy subjects. We initially screened 17 patients with AML, 5 with ALL, 9 with TEL, and 20 healthy subjects. Additionally, 88 AML patients were studied. HeLa, H1, THP1, NB4, and VA13 cell lines were used for functional experiments, and lentivirus vectors containing GFP or RNaseH1-GFP motifs were employed. ATRX, TRF2, TERT, RNaseH1, RNaseH2, TERRA 10q, 15q, 20q, XqYq, XpYp, and 17p expression levels were measured by RT-qPCR. Telomere length was determined by qPCR. R-loops were assessed by immunofluorescence using the S9.6 antibody. Cell line viability was defined by MTT assay. NGS panel was designed to evaluate variants in splicing factors and epigenetic regulators. In the screening population, TERRA expression (10q, 15q, 20q, and XqYq) was significantly increased in AML samples (p < 0.05) in comparison to healthy controls and VA-13 cells. Telomeres were very short in AML samples and TEL patients compared to controls. R-loop formation was significantly increased in AML samples compared to controls (p < 0.01). To examine the TERRA physiology in AML, we further eveluated 88 AML samples. Hierarchical cluster algorithm based on TERRA expression defined two AML groups, one with high and the other with low TERRA expression. Both groups dysplayed short telomeres, but the high TERRA AML group exhibited low TERT and RNaseH2 expression levels (p < 0.05). There was no difference between groups on somatic variants or RNaseH1, TRF2, ATRX, and TRF2 expression levels. AML cell lines were transfected RNaseH1-GFP-containing or empty vector lentiviruses and treated with cytarabine. AML cells with exogenous RNAseH1 expression showed more sensitivity to cytarabine in MTT assays (p < 0.05) than cells transfected with the empty vector. These results show for the first time that TERRA is dysregulated in AML. High TERRA levels are unique to a subset of AML cases with low TERT expression. High TERRA correlated with the accumulation of R-loops, which may be therapeutic targets. TERRA upregulation in AML due to short telomeres is unlikely, given the low TERRA levels in TEL patients. TERRA upregulation did not appear to be directly related to neoplastic differentiation, given low TERRA in ALL. TERRA expression and R-loops are dysregulated in AML. TERRA R-loops inhibition increases chemotherapy-induced apoptosis in AML cell lines and may serve as a therapeutic target.

Methylation of Subtelomeric Chromatin Modifies the Expression of the lncRNA TERRA, Disturbing Telomere Homeostasis.

The long noncoding RNA (lncRNA) telomeric repeat-containing RNA (TERRA) has been associated with telomeric homeostasis, telomerase recruitment, and the process of chromosome healing; nevertheless, the impact of this association has not been investigated during the carcinogenic process. Determining whether changes in TERRA expression are a cause or a consequence of cell transformation is a complex task because studies are usually carried out using either cancerous cells or tumor samples. To determine the role of this lncRNA in cellular aging and chromosome healing, we evaluated telomeric integrity and TERRA expression during the establishment of a clone of untransformed myeloid cells. We found that reduced expression of TERRA disturbed the telomeric homeostasis of certain loci, but the expression of the lncRNA was affected only when the methylation of subtelomeric bivalent chromatin domains was compromised. We conclude that the disruption in TERRA homeostasis is a consequence of cellular transformation and that changes in its expression profile can lead to telomeric and genomic instability.

Expression of Telomeric Repeat-Containing RNA Decreases in Sarcopenia and Increases after Exercise and Nutrition Intervention.

Sarcopenia is defined as aging-related loss of muscle mass and function. Telomere length in chromosomes shortens with age and is modulated by telomeric repeat-containing RNA (TERRA). This study aimed to explore the impact of aging and sarcopenia on telomere length and TERRA expression, and changes following strengthening exercise and nutrition intervention (supplement of branched-chain amino acids, calcium and vitamin D3) for 12 weeks in the sarcopenic population. Older adults (≥65 years old) were divided into non-sarcopenic controls (n = 36) and sarcopenic individuals (n = 36) after measurement of grip strength and body composition. The relative telomere length of leukocytes in all research participants was evaluated using the T/S ratio (telomere/single copy gene), and relative TERRA expression of leukocytes was determined by reverse-transcription qPCR (RT-qPCR). Generalized estimating equation (GEE) was used to analyze the influence of sarcopenia and intervention on the outcomes. There was no significant difference in telomere length between control subjects and participants with sarcopenia. TERRA expression was lower in sarcopenic participants compared to that in non-sarcopenic controls (5.18 ± 2.98 vs. 2.51 ± 1.89; p < 0.001). In the sarcopenic group, intervention significantly increased TERRA expression, but not telomere length. The GEE analysis demonstrated that TERRA expression was negatively associated with sarcopenia (β coefficient = −2.705, p < 0.001) but positively associated with intervention (β coefficient = 1.599, p = 0.023). Sarcopenia is associated with a decrease in TERRA expression in leukocytes. Rebound TERRA expression (returning to the level similar to the non-sarcopenic controls) was observed in the sarcopenic group after exercise and nutrition intervention. Future studies are warranted to examine the potential of TERRA as a biomarker for sarcopenia and its subsequent responses to intervention.

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&amp;lt;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&amp;lt;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

Dynamics of telomeric repeat-containing RNA expression in early embryonic cleavage stages with regards to maternal age.

Telomeres are transcribed into long non-coding RNAs known as Telomeric Repeat-Containing RNA (TERRA). They have been shown to be essential regulators of telomeres and to act as epigenomic modulators at extra-telomeric sites. However the role of TERRA during early embryonic development has never been investigated. Here, we show that TERRA is expressed in murine and bovine early development following a wave pattern. It starts at 4-cell stage, reaching a maximum at the 16-cell followed by a decline at the morula and blastocyst stages. Moreover, TERRA expression is not affected by increasing oocyte donor age whereas telomere length does. This indicates that TERRA expression is independent of the telomere length in early development. Our findings anticipate an essential role of TERRA in early stages of development and this might be useful in the future for a better understanding of age related female infertility.

Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding.

Chromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats – dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA.

Characterization of telomeric repeat-containing RNA (TERRA) localization and protein interactions in primordial germ cells of the mouse†.

Telomeres are dynamic nucleoprotein structures capping the physical ends of linear eukaryotic chromosomes. They consist of telomeric DNA repeats (TTAGGG), the shelterin protein complex, and telomeric repeat-containing RNA (TERRA). Proposed TERRA functions are wide ranging and include telomere maintenance, telomerase inhibition, genomic stability, and alternative lengthening of telomere. However, the presence and role of TERRA in primordial germ cells (PGCs), the embryonic precursors of germ cells, is unknown. Using RNA-fluorescence in situ hybridization, we identify TERRA transcripts in female PGCs at 11.5, 12.5, and 13.5 days postcoitum. In male PGCs, the earliest detection TERRA was at 12.5 dpc where we observed cells with either zero or one TERRA focus. Using qRT-PCR, we evaluated chromosome-specific TERRA expression. Female PGCs showed TERRA expression at 11.5 dpc from eight different chromosome subtelomeric regions (chromosomes 1, 2, 7, 9, 11, 13, 17, and 18) while in male PGCs, TERRA expression was confined to the chromosome 17. Most TERRA transcription in 13.5 dpc male PGCs arose from chromosomes 2 and 6. TERRA interacting proteins were evaluated using identification of direct RNA interacting proteins (iDRiP), which identified 48 in female and 26 in male protein interactors from PGCs at 13.5 dpc. We validated two different proteins: the splicing factor, proline- and glutamine-rich (SFPQ) in PGCs and non-POU domain-containing octamer-binding protein (NONO) in somatic cells. Taken together, our data indicate that TERRA expression and interactome during PGC development are regulated in a dynamic fashion that is dependent on gestational age and sex.

Abstract 2461: Prognostic impact of telomeric repeat containing RNA expression on long-term oncologic outcomes in colorectal cancer

Abstract Purpose: Telomere shortening is associated with colorectal carcinogenesis and transcription from subtelomeres produces telomeric repeat containing RNA (TERRA), a natural inhibitor of telomerase activity (TA). Previous studies on TERRA expression demonstrated downregulation in advanced stages of human tumors. Materials and Methods: We evaluated the 2p TERRA expression and telomere length using quantitative real-time PCR in 60 patients who underwent surgical resection for colorectal cancer between June 2008 and November 2010. Results: Patients were grouped according to 2p TERRA expression, with 27 (45%) and 33 (55%) in the low and high TERRA expression groups, respectively. The median follow-up period was 80 months (range 2-103). 2p TERRA expression was marginally significantly associated with preoperative CEA (P = 0.082) and significantly associated with telomere length (P &amp;lt; 0.05). Five-year overall survival rates in the high 2p TERRA expression group and the low expression group were 73.6% and 78.2%, respectively (p = 0.322), and five-year disease-free survival rates in the high 2p TERRA expression group and the low expression group were 66.1% and 75.1%, respectively (p = 0.317). Conclusions: 2p TERRA expression was marginally significantly associated with preoperative CEA and significantly associated with telomere length; however, it was not prognostic on long-term oncologic outcomes in colorectal cancer. Citation Format: SungUk Bae, Woon Kyung Jeong, Seong Kyu Bae, Jae-Ho Lee. Prognostic impact of telomeric repeat containing RNA expression on long-term oncologic outcomes in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2461.

Regulation of TERRA on telomeric and mitochondrial functions in IPF pathogenesis

BackgroundAging is a known risk factor of idiopathic pulmonary fibrosis (IPF). However, the pathogenic mechanisms underlying the effects of advanced aging remain largely unknown. Telomeric repeat-containing RNA (TERRA) represents a type of long noncoding RNA. In this study, the regulatory roles of TERRA on human telomeres and mitochondria and IPF epithelial injury model were identified.MethodsBlood samples were collected from patients with IPF (n = 24) and matched control individuals (n = 24). The significance of clinical research on the TERRA expression correlated with pulmonary fibrosis was assessed. The expression levels of TERRA in vivo and in vitro were determined through quantitative real-time polymerase chain reaction analysis. Telomerase activity was observed using a fluorescent quantitative TRAP assay kit. The functions of telomeres, mitochondria, and associated genes were analyzed through RNA interference on TERRA.ResultsTERRA expression levels significantly increased in the peripheral blood mononuclear cells of IPF patients. The expression levels also exhibited a direct and significantly inverse correlation with the percentage of predicted force vital capacity, which is a physiological indicator of fibrogenesis during IPF progression. This finding was confirmed in the epithelial injury model of IPF in vitro. RNA interference on TERRA expression can ameliorate the functions of telomeres; mitochondria; associated genes; components associated with telomeres, such as telomerase reverse transcriptase, telomerase, and cell nuclear antigen, cyclin D1; and mitochondria-associated cyclin E genes, including the MMP and Bcl-2 family. The RNA interference on TERRA expression can also improve the functions of oxidative-stress-associated genes, such as reactive oxygen species, superoxide dismutase, and catalase, and apoptosis-related genes, such as cytochrome c, caspase-9, and caspase-3.ConclusionsIn this study, the regulation of TERRA expression on telomeres and mitochondria during IPF pathogenesis was identified for the first time. The results may provide valuable insights for the discovery of a novel biomarker or therapeutic approach for IPF treatment.

Snail1 transcription factor controls telomere transcription and integrity.

Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes.

Telomeric repeat-containing RNA (TERRA) related to polycystic ovary syndrome (PCOS).

Telomeric repeat-containing RNA (TERRA) participates in the regulation of telomere length, and leucocyte telomere length (LTL) plays an important role in the pathophysiology of polycystic ovary syndrome (PCOS), but little is known about the role of TERRA in PCOS. To evaluate the role of TERRA and peripheral blood LTL in PCOS. Forty women with PCOS and 35 healthy women without PCOS were recruited. A prospective case-control study was performed. RNA fluorescence in situ hybridization (FISH) was used to detect TERRA expression in peripheral blood leucocyte. Quantitative PCR was used to measure TERRA expression and the mean LTL in the PCOS and control groups. We analysed the association between related clinical parameters and the age-adjusted ratio of the telomere repeat length (T/S ratio) or TERRA. Telomeric repeat-containing RNA was expressed in human peripheral blood leucocytes, and the signal was abolished after culture with RNase A. The age-adjusted LTLs were significantly longer in the PCOS group than in the control group (P < 0·01). The age-adjusted TERRA level was significantly lower in the PCOS group than in the control group (P < 0·01). Testosterone (TTE) was related positively to LTL and negatively to TERRA in the PCOS group (r = 0·532, P = 0·002; r = -0·477, P = 0·017). We found TERRA expression in human peripheral blood leucocytes, and LTLs were positively associated with PCOS. TERRA and testosterone play an important role in the LTL regulation in PCOS.

Telomere Transcripts Target Telomerase in Human Cancer Cells

Long non-coding transcripts from telomeres, called telomeric repeat-containing RNA (TERRA), were identified as blocking telomerase activity (TA), a telomere maintenance mechanism (TMM), in tumors. We expressed recombinant TERRA transcripts in tumor cell lines with TA and with alternative lengthening of telomeres (ALT) to study effects on TMM and cell growth. Adeno- and lentivirus constructs (AV and LV) were established for transient and stable expression of approximately 130 units of telomere hexanucleotide repeats under control of cytomegalovirus (CMV) and human RNase P RNA H1 (hH1) promoters with and without polyadenylation, respectively. Six human tumor cell lines either using telomerase or ALT were infected and analyzed for TA levels. Pre-infection cells using telomerase had 1%–3% of the TERRA expression levels of ALT cells. AV and LV expression of recombinant TERRA in telomerase positive cells showed a 1.3–2.6 fold increase in TERRA levels, and a decrease in TA of 25%–58%. Dominant-negative or small hairpin RNA (shRNA) viral expression against human telomerase reverse transcriptase (hTERT) results in senescence, not induced by TERRA expression. Population doubling time, cell viability and TL (telomere length) were not impacted by ectopic TERRA expression. Clonal growth was reduced by TERRA expression in TA but not ALT cell lines. ALT cells were not affected by treatments applied. Established cell models and tools may be used to better understand the role of TERRA in the cell, especially for targeting telomerase.

TERRA enhances production of senescence-associated inflammatory cytokines IL-6 and IL-8 (IRC5P.452)

Abstract Aged or senescent cells develop a complex senescence-associated secretory phenotype (SASP). However, the mechanisms underlying the induction of the SASP are unclear. Telomeres are critical for genome stability and aging. Telomeres are transcribed into long non-coding telomeric repeat-containing RNA (TERRA) molecules. We demonstrate that TERRA and inflammatory cytokines were significantly elevated during senescence. Over expression of TERRA significantly upregulated IL-6 and IL-8, but IL-6 and IL-8 did not modulate TERRA expression. The results show that TERRA expression is related to senescence and increases production of SASP. Our findings expand the biological function of TERRA in senscence-assosciated inflammatory responses.

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.

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

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