Telomeric Repeat-containing RNA Research Articles

TERRA-LSD1 phase separation promotes R-loop formation for telomere maintenance in ALT cancer cells

The telomere repeat-containing RNA (TERRA) forms R-loops to promote homology-directed DNA synthesis in the alternative lengthening of telomere (ALT) pathway. Here we report that TERRA contributes to ALT via interacting with the lysine-specific demethylase 1A (LSD1 or KDM1A). We show that LSD1 localizes to ALT telomeres in a TERRA dependent manner and LSD1 function in ALT is largely independent of its demethylase activity. Instead, LSD1 promotes TERRA recruitment to ALT telomeres via RNA binding. In addition, LSD1 and TERRA undergo phase separation, driven by interactions between the RNA binding properties of LSD1 and the G-quadruplex structure of TERRA. Importantly, the formation of TERRA-LSD1 condensates enriches the R-loop stimulating protein Rad51AP1 and increases TERRA-containing R-loops at telomeres. Our findings suggest that LSD1-TERRA phase separation enhances the function of R-loop regulatory molecules for ALT telomere maintenance, providing a mechanism for how the biophysical properties of histone modification enzyme-RNA interactions impact chromatin function.

Unwrap RAP1's Mystery at Kinetoplastid Telomeres.

Although located at the chromosome end, telomeres are an essential chromosome component that helps maintain genome integrity and chromosome stability from protozoa to mammals. The role of telomere proteins in chromosome end protection is conserved, where they suppress various DNA damage response machineries and block nucleolytic degradation of the natural chromosome ends, although the detailed underlying mechanisms are not identical. In addition, the specialized telomere structure exerts a repressive epigenetic effect on expression of genes located at subtelomeres in a number of eukaryotic organisms. This so-called telomeric silencing also affects virulence of a number of microbial pathogens that undergo antigenic variation/phenotypic switching. Telomere proteins, particularly the RAP1 homologs, have been shown to be a key player for telomeric silencing. RAP1 homologs also suppress the expression of Telomere Repeat-containing RNA (TERRA), which is linked to their roles in telomere stability maintenance. The functions of RAP1s in suppressing telomere recombination are largely conserved from kinetoplastids to mammals. However, the underlying mechanisms of RAP1-mediated telomeric silencing have many species-specific features. In this review, I will focus on Trypanosoma brucei RAP1's functions in suppressing telomeric/subtelomeric DNA recombination and in the regulation of monoallelic expression of subtelomere-located major surface antigen genes. Common and unique mechanisms will be compared among RAP1 homologs, and their implications will be discussed.

Long Telomeric Repeat-Containing RNA (TERRA): Biological Functions and Challenges in Vascular Aging and Disease.

Telomere dysfunction is implicated in vascular aging and shorter leucocyte telomeres are associated with an increased risk of atherosclerosis, myocardial infarction, and heart failure. Another pathophysiological mechanism that explains the causal relationship between telomere shortening and atherosclerosis development focuses on the clonal hematopoiesis of indeterminate potential (CHIP), which represents a new and independent risk factor in atherosclerotic cardiovascular diseases. Since telomere attrition has a central role in driving vascular senescence, understanding telomere biology is essential to modulate the deleterious consequences of vascular aging and its cardiovascular disease-related manifestations. Emerging evidence indicates that a class of long noncoding RNAs transcribed at telomeres, known as TERRA for "TElomeric Repeat-containing RNA", actively participates in the mechanisms regulating telomere maintenance and chromosome end protection. However, the multiple biological functions of TERRA remain to be largely elucidated. In particular, the role of TERRA in vascular biology is surprisingly unknown. In this review, we discuss the current knowledge of TERRA and its roles in telomere biology. Additionally, we outline the pieces of evidence that exist regarding the relationship between TERRA dysregulation and disease. Finally, we speculate on how a comprehensive understanding of TERRA transcription in the cardiovascular system may provide valuable insights into telomere-associated vascular aging, offering great potential for new therapeutic approaches.

ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader.

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability. The telomeric repeat-containing RNA (TERRA) that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop. In tumor cells, R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres (ALT) pathway. Dysregulated R-loops can cause stalled replication forks and telomere instability. However, how R-loops are recognized and regulated, particularly at telomeres, is not well understood. We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination. Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses (DDR). In addition, ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway. Using the proximity-dependent biotin identification (BioID) technology, we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases, including DHX9. Importantly, ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9. Our findings suggest that ILF3 may function as a reader of telomeric R-loops, helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

RNA G-quadruplex folding is a multi-pathway process driven by conformational entropy.

The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA by combining all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments. The quadruplex fold is stabilized by cations and thus, the ion atmosphere forming a double layer surrounding the highly charged quadruplex guides the folding process. To capture the ionic double layer in implicit solvent coarse-grained simulations correctly, we develop a matching procedure based on all-atom simulations in explicit water. The procedure yields quantitative agreement between simulations and experiments as judged by the populations of folded and unfolded states at different salt concentrations and temperatures. Subsequently, we show that coarse-grained simulations with a resolution of three interaction sites per nucleotide are well suited to resolve the folding pathways and their intermediate states. The results reveal that the folding progresses from unpaired chain via hairpin, triplex and double-hairpin constellations to the final folded structure. The two- and three-strand intermediates are stabilized by transient Hoogsteen interactions. Each pathway passes through two on-pathway intermediates. We hypothesize that conformational entropy is a hallmark of rG4 folding. Conformational entropy leads to the observed branched multi-pathway folding process for TERRA25. We corroborate this hypothesis by presenting the free energy landscapes and folding pathways of four rG4 systems with varying loop length.

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.

Telomeric repeat-containing RNA is dysregulated in acute myeloid leukemia

AbstractTERRA (telomeric repeat-containing RNA) is a class of long noncoding RNAs transcribed from subtelomeric and telomeric regions. TERRA binds to the subtelomeric and telomeric DNA–forming R-loops (DNA-RNA hybrids), which are involved in telomere maintenance and telomerase function, but the role of TERRA in human cells is not well characterized. Here, we comprehensively investigated for the first time TERRA expression in primary human hematopoietic cells from an exploratory cohort of patients with acute myeloid leukemia (AML), patients with acute lymphoblastic leukemia (ALL), patients with telomere biology disorder (TBD), and healthy subjects. TERRA expression was repressed in primary human hematopoietic cells, including healthy donors, patients with ALL, and patients with TBD, irrespective of their telomere length, except for AML. A second cohort comprising 88 patients with AML showed that TERRA was overexpressed in an AML subgroup also characterized by higher R-loop formation, low TERT and RNAseH2 expression, and a paucity of somatic splicing factor mutations. Telomere length did not correlate with TERRA expression levels. To assess the role of TERRA R-loops in AML, we induced R-loop depletion by increasing RNAseH1 expression in 2 AML cell lines. Decreased TERRA R-loops in AML cell lines resulted in increased chemosensitivity to cytarabine. Our findings indicate that TERRA is uniformly repressed in primary human hematopoietic cells but abnormally expressed in an AML subset with low telomerase.

Exosc9 Initiates SUMO-Dependent lncRNA TERRA Degradation to Impact Telomeric Integrity in Endocrine Therapy Insensitive Hormone Receptor-Positive Breast Cancer.

Long, noncoding RNAs (lncRNAs) are indispensable for normal cell physiology and, consequently, are tightly regulated in human cells. Yet, unlike mRNA, substantially less is known about the mechanisms for lncRNA degradation. It is important to delineate the regulatory control of lncRNA degradation, particularly for lncRNA telomeric repeat-containing RNA (TERRA), as the TERRA-telomere R-loops dictate cell cycle progression and genomic stability. We now report that the exosome complex component Exosc9 degrades lncRNA TERRA in human mammary epithelial cells. Heterochromatin protein 1 alpha (HP1α) recruits Exosc9 to the telomeres; specifically, the SUMO-modified form of HP1α supports interaction with Exosc9 and, as previously reported, lncRNA TERRA. The telomeric enrichment of Exosc9 is cell cycle-dependent and consistent with the loss of telomeric TERRA in the S/G2 phase. Elevated Exosc9 is frequently observed and drives the growth of endocrine therapy-resistant (ET-R) HR+ breast cancer (BCa) cells. Specifically, the knockdown of Exosc9 inversely impacts telomeric R-loops and the integrity of the chromosome ends of ET-R cells. Consistently, Exosc9 levels dictate DNA damage and the sensitivity of ET-R BCa cells to PARP inhibitors. In this regard, Exosc9 may serve as a promising biomarker for predicting the response to PARP inhibitors as a targeted monotherapy for ET-R HR+ BCa.

Differences in Conformational Sampling and Intrinsic Electric Fields Drive Ion Binding in Telomeric and TERRA G-Quadruplexes.

The formation of G-quadruplexes (GQs) occurs in guanine-rich sequences of DNA and RNA, producing highly stable and structurally diverse noncanonical nucleic acid structures. GQs play crucial roles in regulating transcription, translation, and replication and maintaining the genome, among others; thus, changes to their structures can lead to diseases such as cancer. Previous studies using polarizable molecular dynamics simulations have shown differences in ion binding properties between telomeric and telomeric repeat-containing RNA GQs despite architectural similarities. Here, we used volume-based metadynamics and repulsive potential simulations in conjunction with polarizable force fields to quantify the impact of ion binding on the GQ dynamics and ion binding free energies. Furthermore, we describe how GQs exert electric fields on their surroundings to link dynamics with variations in the electronic structure. Our findings provide new insights into the energetic, physical, and conformational properties of GQs and expose subtle but important differences between DNA and RNA GQs with the same fold.

TERRA R-loops connect and protect sister telomeres in mitosis

Resolution of cohesion between sister telomeres in human cells depends on TRF1-mediated recruitment of the polyADP-ribosyltransferase tankyrase to telomeres. In human aged cells, due to insufficient recruitment of TRF1/tankyrase to shortened telomeres, sisters remain cohered in mitosis. This persistent cohesion plays a protective role, but the mechanism by which sisters remain cohered is not well understood. Here we show that telomere repeat-containing RNA (TERRA) holds sister telomeres together through RNA-DNA hybrid (R-loop) structures. We show that a tankyrase-interacting partner, the RNA-binding protein C19orf43, is required for repression of TERRA R-loops. Persistent telomere cohesion in C19orf43-depleted cells is counteracted by RNaseH1, confirming that RNA-DNA hybrids hold sisters together. Consistent with a protective role for persistent telomere cohesion, depletion of C19orf43 in aged cells reduces DNA damage and delays replicative senescence. We propose that the inherent inability of shortened telomeres to recruit R-loop-repressing machinery permits a controlled onset of senescence.

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.

TERRA expression is regulated by the telomere-binding proteins POT-1 and POT-2 in Caenorhabditis elegans.

Several aspects of telomere biology are regulated by the telomeric repeat-containing RNA TERRA. While TERRA expression is conserved through evolution, species-specific mechanisms regulate its biogenesis and function. Here we report on the expression of TERRA in Caenorhabditis elegans. We show that C. elegans TERRA is regulated by the telomere-binding proteins POT-1 and POT-2 which repress TERRA in a telomere-specific manner. C. elegans TERRA transcripts are heterogeneous in length and form discrete nuclear foci, as detected by RNA FISH, in both postmitotic and germline cells; a fraction of TERRA foci localizes to telomeres. Interestingly, in germ cells, TERRA is expressed in all stages of meiotic prophase I, and it increases during pachytene, a stage in meiosis when homologous recombination is ongoing. We used the MS2-GFP system to study the spatiotemporal dynamics of single-telomere TERRA molecules. Single particle tracking revealed different types of motilities, suggesting complex dynamics of TERRA transcripts. Finally, we unveiled distinctive features of C. elegans TERRA, which is regulated by telomere shortening in a telomere-specific manner, and it is upregulated in the telomerase-deficient trt-1; pot-2 double mutant prior to activation of the alternative lengthening mechanism ALT. Interestingly, in these worms TERRA displays distinct dynamics with a higher fraction of fast-moving particles.

TERRA beyond cancer: the biology of telomeric repeat-containing RNAs in somatic and germ cells.

The telomeric noncoding RNA TERRA is a key component of telomeres and it is widely expressed in normal as well as cancer cells. In the last 15years, several publications have shed light on the role of TERRA in telomere homeostasis and cell survival in cancer cells. However, only few studies have investigated the regulation or the functions of TERRA in normal tissues. A better understanding of the biology of TERRA in non-cancer cells may provide unexpected insights into how these lncRNAs are transcribed and operate in cells, and their potential role in physiological processes, such as aging, age-related pathologies, inflammatory processes and human genetic diseases. In this review we aim to discuss the findings that have advanced our understanding of the biology of TERRA using non-cancer mammalian cells as a model system.

The THO complex counteracts TERRA R-loop-mediated telomere fragility in telomerase+ cells and telomeric recombination in ALT+ cells.

Telomeres are the nucleoprotein structures at the ends of linear chromosomes. Telomeres are transcribed into long non-coding Telomeric Repeat-Containing RNA (TERRA), whose functions rely on its ability to associate with telomeric chromatin. The conserved THO complex (THOC) was previously identified at human telomeres. It links transcription with RNA processing, decreasing the accumulation of co-transcriptional DNA:RNA hybrids throughout the genome. Here, we explore the role of THOC at human telomeres, as a regulator of TERRA localization to chromosome ends. We show that THOC counteracts TERRA association with telomeres via R-loops formed co-transcriptionally and also post-transcriptionally, in trans. We demonstrate that THOC binds nucleoplasmic TERRA, and that RNaseH1 loss, which increases telomeric R-loops, promotes THOC occupancy at telomeres. Additionally, we show that THOC counteracts lagging and mainly leading strand telomere fragility, suggesting that TERRA R-loops can interfere with replication fork progression. Finally, we observed that THOC suppresses telomeric sister-chromatid exchange and C-circle accumulation in ALT cancer cells, which maintain telomeres by recombination. Altogether, our findings reveal crucial roles of THOC in telomeric homeostasis through the co- and post-transcriptional regulation of TERRA R-loops.

The exoribonuclease XRN2 mediates degradation of the long non-coding telomeric RNA TERRA.

The telomeric repeat-containing RNA, TERRA, associates with both telomeric DNA and telomeric proteins, often forming RNA:DNA hybrids (R-loops). TERRA is most abundant in cancer cells utilizing the alternative lengthening of telomeres (ALT) pathway for telomere maintenance, suggesting that persistent TERRA R-loops may contribute to activation of the ALT mechanism. Therefore, we sought to identify the enzyme(s) that regulate TERRA metabolism in mammalian cells. Here, we identify that the 5'-3' exoribonuclease XRN2 regulates the stability of TERRA RNA. Moreover, while stabilization of TERRA alone was insufficient to drive ALT, depletion of XRN2 in ALT-positive cells led to a significant increase in TERRA R-loops and exacerbated ALT activity. Together, our findings highlight XRN2 as a key determinant of TERRA metabolism and telomere stability in cancer cells that rely on the ALT pathway.

Excited-State Charge Transfer, Proton Transfer, and Exciplex Formation Revealed by Ultrafast Time-Resolved Spectroscopy in Human Telomeric Ribonucleic Acid Quadruplex.

Guanine quadruplexes (GQs), important for genome stability and biotechnology application, can form from both DNA and RNA. However, unlike the study of DNA GQs, little research has been conducted on excited states of GQs from RNA, which due to the ribose 2'-hydroxy group have structures distinct from their DNA counterparts. Combining ultrafast broadband time-resolved fluorescence and transient absorption measurements, we report the first direct probe of excitation dynamics for a bimolecular GQ from human telomeric repeat-containing RNA taking the typical highly compacted parallel folding with a propeller-like loop structure. The result revealed a multichannel decay containing an unusual high-energy excimer featuring charge transfer deactivated by rapid proton transfer in the tetrad core region. It also identified an unprecedented exciplex displaying massively red-shifted fluorescence produced from charge transfer in the loop region. The findings underscore the role of structural conformation and base content in determining the energy, electronic attribution, and decay dynamics of GQ excited states.

Translating the telomeres

Telomeres are transcribed into long noncoding telomeric repeat-containing RNA (TERRA). Or so we thought. Recently, Al-Turki and Griffith provided evidence that TERRA can code for valine-arginine (VR) or glycine-leucine (GL) dipeptide repeat proteins by undergoing repeat-associated non-ATG (RAN) translation. This finding uncovers a new mechanism by which telomeres can impact cellular function.

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.

A Randomized Controlled Intervention Trial with Danazol to Improve Telomeric and Fertility Parameters in Women with Diminished Ovarian Reserve: A Pilot Study.

Most women who are treated at in vitro fertilization (IVF) clinics have trouble conceiving due to ovarian failure (OF), which seems to be associated to short telomeres and reduced or absent telomerase activity in their granulosa cells. Indeed, telomere pathways are involved in organ dysfunction. However, sexual steroids can stimulate the expression of the telomerase gene and have been successfully used to prevent telomere attrition. Thus, a strategy to improve IVF outcomes in women with OF could be telomerase reactivation using sexual steroids. We conducted a double-blind, placebo-controlled study. Patients with diminished ovarian reserve were randomized to Danazol or placebo for 3 months. We included patients with normal ovarian reserve in the study as untreated controls. Patients and controls underwent several ovarian stimulations (OSs). Telomere and IVF parameters were assessed. We found that the mean telomere length in blood and the percentage of short and long telomeres were similar throughout the 3 months of treatment with Danazol. Remarkably, while the number of cells with one telomeric repeat-containing RNA (TERRA) focus decreased (p = 0.04) after the first month of Danazol treatment, the number of cells with 2 to 4 TERRA foci increased (p = 0.02). Regarding fertility, no differences were found in the antral follicle count. Interestingly, in OS performed after the trial, all Danazol-treated patients had a better MII oocyte rate compared to OS performed before the pilot study.EudraCT number: 2018-004400-19. Danazol treatment seemed to affect telomere maintenance, since both the number of TERRA foci and the ratio of MII oocytes changed. However, further research is needed to confirm these results.

Genome-wide gene expression responses to experimental manipulation of Saccharomyces cerevisiae repressor activator protein 1 (Rap1) expression level

Precise regulation of transcription in gene expression is critical for all aspects of normal organism form, fitness, and function and even minor alterations in the level, location, and timing of gene expression can result in phenotypic variation within and between species including evolutionary innovations and human disease states. Eukaryotic transcription is regulated by a complex interplay of multiple factors working both at a physical and molecular levels influencing this process. In Saccharomyces cerevisiae, the TF with the greatest number of putative regulatory targets is the essential gene Repressor Activator Protein 1 (RAP1). While much is known about the roles of Rap1 in gene regulation and numerous cellular processes, the response of Rap1 target genes to systematic titration of RAP1 expression level remains unknown. To fill this knowledge gap, we used a strain with a tetracycline-titratable promoter replacing wild-type regulatory sequences of RAP1 to systematically reduce the expression level of RAP1 and followed this with RNA sequencing (RNA-seq) to measure genome-wide gene expression responses. Previous research indicated that Rap1 plays a significant regulatory role in particular groups of genes including telomere-proximal genes, homothallic mating (HM) loci, glycolytic genes, DNA repair genes, and ribosomal protein genes; therefore, we focused our analyses on these groups and downstream targets to determine how they respond to reductions in RAP1 expression level. Overall, despite being known as both an activator and as a repressor of its target genes, we found that Rap1 acts as an activator for more target genes than as a repressor. Additionally, we found that Rap1 functions as an activator of ribosomal protein genes and a repressor for HM loci genes consistent with predictions from the literature. Unexpectedly, we found that Rap1 functions as a repressor of glycolytic enzyme genes contrary to prior reports of it having the opposite effect. We also compared the expression of RAP1 to five different genes related to DNA repair pathway and found that decreasing RAP1 downregulated four of those five genes. Finally, we found no effect of RAP1 depletion on telomere-proximal genes despite its functioning to silence telomeric repeat-containing RNAs. Together our results enrich our understanding of this important transcriptional regulator.