Telomeric Repeat-containing RNA Research Articles

Effects of cation charges on the binding of stabilizers with human telomere and TERRA G-quadruplexes

Both telomere and telomeric repeat-containing RNAs (TERRA) can fold into G-quadruplexes (G4) in eukaryotic cells. Given their key roles in the regulation of telomere length and translation, telomere and TERRA G4 are interesting targets of novel drug development strategies. It is known that the cation charge of a stabilizer is crucial to the binding of G4 and stabilizer. However, the quantitative relationship between the cation charge of a stabilizer and the binding strengths with telomere and TERRA G4 remain unclear. In the current study, by substituting positive charged TMPyP4 with neutral and negative charged groups, the effects of cation charges on the binding conformation and binding strength of porphyrin stabilizers are investigated via molecular docking and molecular dynamic (MD) simulations. The results show that all TMPyP4 analogs form stable binding complexes with telomere and TERRA G4 and that, stabilizer charges have limited effects on binding conformation and can hardly lead to any special conformational alternations of G4. Our hydrogen bond analysis shows that all stabilizers can hardly form stable intermolecular hydrogen bonds with G4. Regarding binding strength levels, a linear correlation is found between the binding free energies and cation charges of stabilizers in all G4‒stabilizer complexes, revealing the pivotal role of electrostatic interactions. The present work is the first to reveal a quantitative correlation between the charges and binding strengths of stabilizers in their binding with human telomere and TERRA G4, which will prove pivotal for G4 targeted drug design and development.

G-quadruplex binding ability of TLS/FUS depends on the β-spiral structure of the RGG domain.

The RGG domain, defined as closely spaced Arg-Gly-Gly repeats, is a DNA and RNA-binding domain in various nucleic acid-binding proteins. Translocated in liposarcoma (TLS), which is also called FUS, is a protein with three RGG domains, RGG1, RGG2 and RGG3. TLS/FUS binding to G-quadruplex telomere DNA and telomeric repeat-containing RNA depends especially on RGG3, comprising Arg-Gly-Gly repeats with proline- and arginine-rich regions. So far, however, only non-specific DNA and RNA binding of TLS/FUS purified with buffers containing urea and KCl have been reported. Here, we demonstrate that protein purification using a buffer with high concentrations of urea and KCl decreases the G-quadruplex binding abilities of TLS/FUS and RGG3, and disrupts the β-spiral structure of RGG3. Moreover, the Arg-Gly-Gly repeat region in RGG3 by itself cannot form a stable β-spiral structure that binds to the G-quadruplex, because the proline- and arginine-rich regions induce the β-spiral structure and the G-quadruplex-binding ability of RGG3. Our findings suggest that the G-quadruplex-specific binding abilities of TLS/FUS require RGG3 with a β-spiral structure stabilized by adjacent proline- and arginine-regions.

Human telomere RNA structure and function

A recent finding demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA (referred to as TERRA) in mammalian cells. The existence of TERRA RNA may reveal a new level of regulation and protection of chromosome ends that could promote valuable insight into fundamental biological processes such as cancer and aging. Revealing the structure and function of telomere RNA will be essential for understanding telomere biology and telomere-related diseases. In fact, others and we have shown by NMR and X-ray crystallography that human telomere RNA forms G-quadruplex structures. We found that human telomere RNA forms a G-quadruplex dimer in the living cells by employing a light-switching probe. Recently, we also demonstrated that telomere RNA G-quadruplex structures play an important role in providing a protective structure for telomere ends. This review highlights the structures and topologies for telomere RNA G-quadruplex and recent efforts in the understanding of telomere RNA function; outlines the future challenges in the field.

DNA:RNA hybrids at telomeres - when it is better to be out of the (R) loop.

R-loops (RLs) are three-stranded nucleic acid structures that contain a DNA:RNA hybrid and a displaced DNA strand. Genomic regions with GC skew and a G-rich transcript are particularly prone to form RLs. RLs play important physiological roles in cells; however, when present at abnormally high levels, they may threaten genome stability. The perfect GC skew of telomeric repeats and the discovery of telomeric repeat-containing RNA (TERRA), a long noncoding transcript that consists of the G-rich telomeric sequence, make telomeric sequences the perfect candidates for generating RLs. Indeed, in the past 5years, telomere R-loops (TRLs) have been demonstrated in Saccharomycescerevisiae, Trypanosomabrucei, and human cells. The presence of TRLs in normal human cells that transcribe low levels of TERRA, suggests a physiological role for these nucleic structures in telomere maintenance. Abnormally enhanced TERRA transcription, as found in several human pathological conditions, leads to high TRL levels and various cellular outcomes, depending on the recombinogenic capabilities of the cells. Study of TRLs in various organisms highlights the necessity for tight regulation of these structures, which can switch from beneficial to detrimental under different conditions. Here, we review the current state of knowledge on TRLs, describe several means by which TRLs are regulated, and discuss how findings from yeast are relevant to human pathological scenarios in which TRLs are deregulated.

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

ABSTRACTTelomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

Inflammatory factor receptor Toll-like receptor 4 controls telomeres through heterochromatin protein 1 isoforms in liver cancer stem cell.

Toll‐like receptor 4 (TLR4) which acts as a receptor for lipopolysaccharide (LPS) has been reported to be involved in carcinogenesis. However, the regulatory mechanism of it has not been elucidated. Herein, we demonstrate that TLR4 promotes the malignant growth of liver cancer stem cells. Mechanistically, TLR4 promotes the expression of histone‐lysine N‐methyltransferase (SUV39 h2) and increases the formation of trimethyl histone H3 lysine 9‐heterochromatin protein 1‐telomere repeat binding factor 2 (H3K9me3‐HP1‐TRF2) complex at the telomeric locus under mediation by long non coding RNA urothelial cancer‐associated 1 (CUDR). At the telomeric locus, this complex promotes binding of POT1, pPOT1, Exo1, pExo1, SNM1B and pSNM1B but prevents binding of CST/AAF to telomere, thus controlling telomere and maintaining telomere length. Furthermore, TLR4 enhances interaction between HP1α and DNA methyltransferase (DNMT3b), which limits RNA polymerase II deposition on the telomeric repeat‐containing RNA (TERRA) promoter region and its elongation, thus inhibiting transcription of TERRA. Ultimately, TLR4 enhances the telomerase activity by reducing the interplay between telomerase reverse transcriptase catalytic subunit (TERT) and TERRA. More importantly, our results reveal that tri‐complexes of HP1 isoforms (α, β and γ) are required for the oncogenic action of TLR4. This study elucidates a novel protection mechanism of TLR4 in liver cancer stem cells and suggests that TLR4 can be used as a novel therapeutic target for liver cancer.

Induction and relocalization of telomeric repeat-containing RNAs during diauxic shift in budding yeast.

Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase. In the budding yeast Saccharomyces cerevisiae, TERRA accumulation is repressed by telomeric silencing and the Rat1 exonuclease. On the other hand, telomere shortening promotes expression of TERRA. So far, little is known about the biological processes that induce TERRA expression in yeast. Understanding the dynamics of TERRA expression and localization is essential to define its function in telomere biology. Here, we aim to study the dynamics of TERRA expression during yeast cell growth. Using live-cell imaging, RNA-FISH and quantitative RT-PCR, we show that TERRA expression is induced as yeast cells undergo diauxic shift, a lag phase during which yeast cells switch their metabolism from anaerobic fermentation to oxidative respiration. This induction is transient as TERRA levels decrease during post-diauxic shift. The increased expression of TERRA is not due to the shortening of telomeres or increased stability of this transcript. Surprisingly, this induction is coincident with a cytoplasmic accumulation of TERRA molecules. Our results suggest that TERRA transcripts may play extranuclear functions with important implications in telomere biology and add a novel layer of complexity in the interplay between telomere biology, metabolism and stress response.

Increased TERRA levels and RNase H sensitivity are conserved hallmarks of post-senescent survivors in budding yeast

Cancer cells activate telomere maintenance mechanisms (TMMs) to bypass replicative senescence and achieve immortality by either upregulating telomerase or promoting homology-directed repair (HDR) at chromosome ends to maintain telomere length, the latter being referred to as ALT (Alternative Lengthening of Telomeres). In yeast telomerase mutants, the HDR-based repair of telomeres leads to the generation of ‘survivors’ that escape senescence and divide indefinitely. So far, yeast has proven to provide an accurate model to study the generation and maintenance of telomeres via HDR. Recently, it has been established that up-regulation of the lncRNA, TERRA (telomeric repeat-containing RNA), is a novel hallmark of ALT cells. Moreover, RNA-DNA hybrids are thought to trigger HDR at telomeres in ALT cells to maintain telomere length and function. Here we show that, also in established yeast type II survivors, TERRA levels are increased in an analogous manner to human ALT cells. The elevated TERRA levels are independent of yeast-specific subtelomeric structures, i.e. the presence or absence of Y’ repetitive elements. Furthermore, we show that RNase H1 overexpression, which degrades the RNA moiety in RNA-DNA hybrids, impairs the growth of yeast survivors. We suggest that even in terms of TERRA regulation, yeast survivors serve as an accurate model that recapitulates many key features of human ALT cells.

Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA

The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA. We then showed that the third RGG motif of TLS, the RGG3 domain, is responsible for the complex formation. However, the structural basis for their binding remains obscure. Here, NMR-based binding assaying revealed the interactions in the binary and ternary complexes of RGG3 with telomeric DNA or/and TERRA. In the ternary complex, tyrosine bound exclusively to TERRA, while phenylalanine bound exclusively to telomeric DNA. Thus, tyrosine and phenylalanine each play a central role in the recognition of TERRA and telomeric DNA, respectively. Surprisingly in the binary complexes, RGG3 used both tyrosine and phenylalanine residues to bind to either TERRA or telomeric DNA. We propose that the plastic roles of tyrosine and phenylalanine are important for RGG3 to efficiently form the ternary complex, and thereby regulate the telomere shortening.

Long-term Persistent Organic Pollutants Exposure Induced Telomere Dysfunction and Senescence-Associated Secretary Phenotype.

Environmentally persistent organic pollutant (POP) is the general term for refractory organic compounds that show long-range atmospheric transport, environmental persistence, and bioaccumulation. It has been reported that the accumulation of POPs could lead to cellular DNA damage and adverse effects of on metabolic health. To better understand the mechanism of the health risks associated with POPs, we conducted an evidence-based cohort investigation (n = 5,955) at the Jinghai e-waste disposal center in China from 2009 to 2016, where people endure serious POP exposure. And high levels of aging-related diseases, including hypertension, diabetes, autoimmune diseases, and reproductive disorders were identified associated with the POP exposure. In the subsequent molecular level study, an increased telomere dysfunction including telomere multiple telomere signals, telomere signal-free ends, telomere shortening and activation of alternative lengthening of telomeres were observed, which might result from the hypomethylated DNA modification induced telomeric repeat-containing RNA overexpression. Moreover, dysfunctional telomere-leaded senescence-associated secretory phenotype was confirmed, as the proinflammatory cytokines and immunosenescence hallmarks including interleukin-6, P16INK4a, and P14ARF were stimulated. Thus, we proposed that the dysfunctional telomere and elevated systemic chronic inflammation contribute to the aging-associated diseases, which were highly developed among the POP exposure individuals.

Telomeric Repeat-Containing RNAs (TERRA) Decrease in Squamous Cell Carcinoma of the Head and Neck Is Associated with Worsened Clinical Outcome.

Telomeres are transcribed into noncoding telomeric repeat-containing RNAs (TERRA), which are essential for telomere maintenance. Deregulation of TERRA transcription impairs telomere metabolism and a role in tumorigenesis has been proposed. Head and neck cancer (HNC) is one of the most frequent cancers worldwide, with head and neck squamous cell carcinoma (HNSCC) being the predominant type. Since HNSCC patients are characterized by altered telomere maintenance, a dysfunction in telomere transcription can be hypothesized. In this prospective study, we compared TERRA levels in the tumor and matched normal tissue from 23 HNSCC patients. We then classified patients in two categories according to the level of TERRA expression in the tumor compared to the normal tissue: (1) lower expression in the tumor, (2) higher or similar expression in tumor. A significant proportion of patients in the first group died of the disease within less than 34 months postsurgery, while the majority of patients in the second group were alive and disease-free. Our results highlight a striking correlation between TERRA expression and tumor aggressiveness in HNSCC suggesting that TERRA levels may be proposed as a novel molecular prognostic marker for HNSCC.

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.

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.

Eroded telomeres are rearranged in quiescent fission yeast cells through duplications of subtelomeric sequences

While the mechanisms of telomere maintenance has been investigated in dividing cells, little is known about the stability of telomeres in quiescent cells and how dysfunctional telomeres are processed in non-proliferating cells. Here we examine the stability of telomeres in quiescent cells using fission yeast. While wild type telomeres are stable in quiescence, we observe that eroded telomeres were highly rearranged during quiescence in telomerase minus cells. These rearrangements depend on homologous recombination (HR) and correspond to duplications of subtelomeric regions. HR is initiated at newly identified subtelomeric homologous repeated sequences (HRS). We further show that TERRA (Telomeric Repeat-containing RNA) is increased in post-mitotic cells with short telomeres and correlates with telomere rearrangements. Finally, we demonstrate that rearranged telomeres prevent cells to exit properly from quiescence. Taken together, we describe in fission yeast a mode of telomere repair mechanism specific to post-mitotic cells that is likely promoted by transcription.

Paf1 and Ctr9, core components of the PAF1 complex, maintain low levels of telomeric repeat containing RNA.

The conserved PAF1 complex (Cdc73, Paf1, Ctr9, Leo1 and Rtf1, in yeast), binds RNA pol II, and affects levels of many RNAs. Although PAF1 is a complex, there is evidence that different components perform different functions. In yeast, Cdc73, Paf1 and Ctr9 maintain normal telomerase RNA (TLC1) levels and affect telomere length. Here we report a new connection between the PAF1 complex and telomere biology. We show that Paf1 and Ctr9 maintain low telomere repeat containing RNA (TERRA) levels while Cdc73, Leo1 and Rtf1 have lesser effects. Analysis of double mutants shows that Paf1 and Ctr9 can affect TERRA independently of Sir4, Rat1, and Trf4, previously identified regulators of TERRA. The data suggest that Paf1 and Ctr9 maintain low TERRA levels by affecting both transcription and degradation and that short telomeres in cdc73Δ, paf1Δ and ctr9Δ mutants do not induce TERRA. These data establish the PAF1 complex as a new regulator of TERRA, and are consistent with the model in which Paf1 and Ctr9, the core components of the PAF1 complex, affect transcript levels and cell fitness by numerous mechanisms.

Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation.

Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

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) is activated during early mouse development, from 2-cell to blastocyst stages, in a cell cycle-dependent manner

Telomeric repeat-containing RNA (TERRA) is activated during early mouse development, from 2-cell to blastocyst stages, in a cell cycle-dependent manner

The Telomeric Response to Viral Infection.

The ends of linear genomes, whether viral or cellular, can elicit potent DNA damage and innate immune signals. DNA viruses entering the nucleus share many features with telomeres in their ability to either suppress or co-opt these pathways. Here, we review some of the common mechanisms that viruses and telomeres use to manage the DNA damage and innate immune response pathways. We highlight recent studies on the role of the telomere repeat-containing RNA (TERRA) in response to viral infection. We discuss how TERRA can be activated through a p53-response element embedded in a retrotransposon-like repeat found in human subtelomeres. We consider how TERRA can function as a danger signal when secreted in extracellular vesicles to induce inflammatory cytokines in neighboring cells. These findings suggest that TERRA may be part of the innate immune response to viral infection, and support the hypothesis that telomeres and viruses utilize common mechanisms to maintain genome integrity and regulate innate immunity.

Role of telomeric repeat-containing RNA in telomeric chromatin remodeling during the early expansion of human embryonic stem cells.

This study aimed to explore the role of telomeric repeat-containing RNA (TERRA) in telomeric chromatin remodeling during the early expansion of human embryonic stem cells (hESCs). During the derivation of hESCs, histone demethylation in the telomeric region facilitates telomerase-mediated telomere elongation. An adequate telomere repeat is essential for hESCs to acquire and/or maintain the unlimited symmetric division, which suggests that there is a link between pluripotency and telomere maintenance. The present study found that the gradual decrease in TERRA levels and related TERRA foci were correlated with telomeric length elongation in the early expansion of hESCs. In addition, TERRA participated in telomeric chromatin remodeling by cooperating with SUV39H1 (suppressor of variegation 3-9 homolog 1/2) to propagate telomeric heterochromatin marker, histone H3 trimethylation of lysine 9. Moreover, the fibroblast growth factor signaling pathway, which is activated in hESCs, could suppress TERRA levels via telomeric repeat factor 1, which results in reduced SUV39H1 recruitment by TERRA at the telomere. Taken together, these results highlight the role of TERRA in hESC telomere elongation and homeostasis in the acquisition and/or maintenance of stem cell pluripotency.-Zeng, S., Liu, L., Sun, Y., Lu, G., Lin, G. Role of telomeric repeat-containing RNA in telomeric chromatin remodeling during the early expansion of human embryonic stem cells.