Separation of telomere protection from length regulation by two different point mutations at amino acid 492 of RTEL1.

RTEL1 is an essential DNA helicase that plays multiple roles in genome stability and telomere length regulation. A variant of RTEL1 with a lysine at position 492 is associated with short telomeres in Mus spretus , while a conserved methionine at this position is found in M. musculus, which has ultra-long telomeres. In humans, a missense mutation at this position ( RTEL1 M492I ) causes a fatal telomere biology disease termed Hoyeraal-Hreidarsson syndrome (HHS). We previously described a M. musculus mouse model termed 'Telomouse', in which changing methionine 492 to a lysine (M492K) shortened the telomeres to their length in humans. Here, we report on the derivation of a mouse strain carrying the M492I mutation, termed 'HHS mouse'. The HHS mouse telomeres are not as short as those of Telomice but nevertheless they display higher levels of telomeric DNA damage, fragility and recombination, associated with anaphase bridges and micronuclei. These observations indicate that the two mutations separate critical functions of RTEL1: M492K mainly reduces the telomere length setpoint, while M492I predominantly disrupts telomere protection. The two mouse models enable dissecting the mechanistic roles of RTEL1 and the different contributions of short telomeres and DNA damage to telomere biology diseases, genomic instability, cancer, and aging.

The many faces of the helicase RTEL1 at telomeres and beyond.

Regulator of telomere elongation 1 (RTEL1) is known as a DNA helicase that is important for telomeres and genome integrity. However, the diverse phenotypes of RTEL1 dysfunction, the wide spectrum of symptoms caused by germline RTEL1 mutations, and the association of RTEL1 mutations with cancers suggest that RTEL1 is a complex machine that interacts with DNA, RNA, and proteins, and functions in diverse cellular pathways. We summarize the proposed functions of RTEL1 and discuss their implications for telomere maintenance. Studying RTEL1 is crucial for understanding the complex interplay between telomere maintenance and other nuclear pathways, and how compromising these pathways causes telomere biology diseases, various aging-associated pathologies, and cancer.

Telomouse-a mouse model with human-length telomeres generated by a single amino acid change in RTEL1.

Telomeres, the ends of eukaryotic chromosomes, protect genome integrity and enable cell proliferation. Maintaining optimal telomere length in the germline and throughout life limits the risk of cancer and enables healthy aging. Telomeres in the house mouse, Mus musculus, are about five times longer than human telomeres, limiting the use of this common laboratory animal for studying the contribution of telomere biology to aging and cancer. We identified a key amino acid variation in the helicase RTEL1, naturally occurring in the short-telomere mouse species M. spretus. Introducing this variation into M. musculus is sufficient to reduce the telomere length set point in the germline and generate mice with human-length telomeres. While these mice are fertile and appear healthy, the regenerative capacity of their colonic epithelium is compromised. The engineered Telomouse reported here demonstrates a dominant role of RTEL1 in telomere length regulation and provides a unique model for aging and cancer.

Accelerated replicative senescence of ataxia-telangiectasia skin fibroblasts is retained at physiologic oxygen levels, with unique and common transcriptional patterns.

The genetic disorder, ataxia-telangiectasia (A-T), is caused by loss of the homeostatic protein kinase, ATM, and combines genome instability, tissue degeneration, cancer predisposition, and premature aging. Primary fibroblasts from A-T patients exhibit premature senescence when grown at ambient oxygen concentration (21%). Here, we show that reducing oxygen concentration to a physiological level range (3%) dramatically extends the proliferative lifespan of human A-T skin fibroblasts. However, they still undergo senescence earlier than control cells grown under the same conditions and exhibit high genome instability. Comparative RNA-seq analysis of A-T and control fibroblasts cultured at 3% oxygen followed by cluster analysis of differentially expressed genes and functional enrichment analysis, revealed distinct transcriptional dynamics in A-T fibroblasts senescing in physiological oxygen concentration. While some transcriptional patterns were similar to those observed during replicative senescence of control cells, others were unique to the senescing A-T cells. We observed in them a robust activation of interferon-stimulated genes, with undetected expression the interferon genes themselves. This finding suggests an activation of a non-canonical cGAS-STING-mediated pathway, which presumably responds to cytosolic DNA emanating from extranuclear micronuclei detected in these cells. Senescing A-T fibroblasts also exhibited a marked, intriguely complex alteration in the expression of genes associated with extracellular matrix (ECM) remodeling. Notably, many of the induced ECM genes encode senescence-associated secretory phenotype (SASP) factors known for their paracrine pro-fibrotic effects. Our data provide a molecular dimension to the segmental premature aging observed in A-T patients and its associated symptoms, which develop as the patients advance in age.

Functional Interactions of Kluyveromyces lactis Telomerase Reverse Transcriptase with the Three-Way Junction and the Template Domains of Telomerase RNA.

The ribonucleoprotein telomerase contains two essential components: telomerase RNA (TER) and telomerase reverse transcriptase (TERT, Est2 in yeast). A small portion of TER, termed the template, is copied by TERT onto the chromosome ends, thus compensating for sequence loss due to incomplete DNA replication and nuclease action. Although telomerase RNA is highly divergent in sequence and length across fungi and mammals, structural motifs essential for telomerase function are conserved. Here, we show that Est2 from the budding yeast Kluyveromyces lactis (klEst2) binds specifically to an essential three-way junction (TWJ) structure in K. lactis TER, which shares a conserved structure and sequence features with the essential CR4-CR5 domain of vertebrate telomerase RNA. klEst2 also binds specifically to the template domain, independently and mutually exclusive of its interaction with TWJ. Furthermore, we present the high-resolution structure of the klEst2 telomerase RNA-binding domain (klTRBD). Mutations introduced in vivo in klTRBD based on the solved structure or in TWJ based on its predicted RNA structure caused severe telomere shortening. These results demonstrate the conservation and importance of these domains and the multiple protein-RNA interactions between Est2 and TER for telomerase function.

Leukocyte Telomere Length Correlates with Extended Female Fertility.

Current social trends of delayed reproduction to the fourth and fifth decade of life call for a better understanding of reproductive aging. Demographic studies correlated late reproduction with general health and longevity. Telomeres, the protective ends of eukaryotic chromosomes, were implicated in various aging-associated pathologies and longevity. To examine whether telomeres are also associated with reproductive aging, we measured by Southern analysis the terminal restriction fragments (TRF) in leukocytes of women delivering a healthy infant following a spontaneous pregnancy at 43-48 years of age. We compared them to age-matched previously fertile women who failed to conceive above age 41. The average TRF length in the extended fertility group (9350 bp) was significantly longer than in the normal fertility group (8850 bp; p-value = 0.03). Strikingly, excluding women with nine or more children increased the difference between the groups to over 1000 bp (9920 and 8880 bp; p-value = 0.0009). Nevertheless, we observed no apparent effects of pregnancy, delivery, or parity on telomere length. We propose that longer leukocyte telomere length reflects higher oocyte quality, which can compensate for other limiting physiological and behavioral factors and enable successful reproduction. Leukocyte telomere length should be further explored as a novel biomarker of oocyte quality for assessing reproductive potential and integrating family planning with demanding women's careers.

Inherited human Apollo deficiency causes severe bone marrow failure and developmental defects.

Inherited bone marrow failure syndromes (IBMFSs) are a group of disorders typified by impaired production of 1 or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant, Høyeraal-Hreidarsson (HH) syndrome, are rare IBMFSs characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. We identified biallelic variants in the gene encoding the 5'-to-3' DNA exonuclease Apollo/SNM1B in 3 unrelated patients presenting with a DC/HH phenotype consisting of early-onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly, and/or intrauterine growth retardation. All 3 patients carry a homozygous or compound heterozygous (in combination with a null allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients' cells showed signs of telomere fragility that were not associated with global reduction of telomere length. Unlike patients' cells, human Apollo KO HT1080 cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS that combines clinical hallmarks of DC/HH with normal telomere length.

Telomerase, the recombination machinery and Rap1 play redundant roles in yeast telomere protection.

Telomeres are specialized nucleoprotein complexes that protect the ends of eukaryotic chromosomes and distinguish them from broken DNA ends. Disruption of telomere protection may cause aging-associated pathologies and cancer. Here, we examined what makes telomere protection durable and resistant to perturbations using a budding yeast model organism. The protein Rap1 binds the telomeric repeats, negatively regulates telomere length, and protects telomeres by repressing homologous recombination and non-homologous end joining (NHEJ). A single-nucleotide mutation in the Kluyveromyces lactis telomerase RNA (TER1) template, ter1-16T, is incorporated into the telomeric repeats, disrupting the binding of Rap1 and causing dramatic telomere elongation. However, cell viability is not significantly affected, suggesting the existence of additional mechanism(s) for telomere protection. To examine this hypothesis, we explored the contribution of the recombination factor Rad52 and telomerase to telomere protection in the background of ter1-16T. To disrupt the function of telomerase, we exploited small mutations in a stem-loop domain of TER1 (Reg2), which result in short but stable telomeres. We generated K. lactis strains with combinations of three different mutations: ter1-16T, RAD52 deletion, and a two-nucleotide substitution in Reg2. Our results show that upon Rap1 depletion from telomeres, telomerase and the recombination machinery compensate for the loss of Rap1 protection and play redundant but critical roles in preventing NHEJ and maintaining telomere integrity and cell viability. These results demonstrate how redundant pathways make the essential role of telomeres-protecting our genome integrity and preventing cancer-more robust and resistant to assaults and perturbations.

An RTEL1 Mutation Links to Infantile-Onset Ulcerative Colitis and Severe Immunodeficiency.

PURPOSE: More than 50 different monogenic disorders causing inflammatory bowel disease (IBD) have been identified. Our goal was to characterize the clinical phenotype, genetic workup, and immunologic alterations in an Ashkenazi Jewish patient that presented during infancy with ulcerative colitis and unique clinical manifestations. METHODS: Immune workup and whole-exome sequencing were performed, along with Sanger sequencing for confirmation. Next-generation sequencing of the TCRB and IgH was conducted for immune repertoire analysis. Telomere length was evaluated by in-gel hybridization assay. Mass cytometry was performed on patient's peripheral blood mononuclear cells, and compared with control subjects and patients with UC. RESULTS: The patient presented in infancy with failure to thrive and dysmorphic features, consistent with a diagnosis of dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome. Severe ulcerative colitis manifested in the first year of life and proceeded to the development of a primary immunodeficiency, presenting as Pneumocystis jiroveci pneumonia and hypogammaglobulinemia. Genetic studies identified a deleterious homozygous C.3791G>A missense mutation in the helicase regulator of telomere elongation 1 (RTEL1), leading to short telomeres in the index patient. Immune repertoire studies showed polyclonal T and B cell receptor distribution, while mass cytometry analysis demonstrated marked immunological alterations, including a predominance of naïve T cells, paucity of B cells, and a decrease in various innate immune subsets. CONCLUSIONS: RTEL1 mutations are associated with significant alterations in immune landscape and can manifest with infantile-onset IBD. A high index of suspicion is required in Ashkenazi Jewish families where the carriage rate of the C.3791G>A variant is high.

Full length RTEL1 is required for the elongation of the single-stranded telomeric overhang by telomerase.

Telomeres cap the ends of eukaryotic chromosomes and distinguish them from broken DNA ends to suppress DNA damage response, cell cycle arrest and genomic instability. Telomeres are elongated by telomerase to compensate for incomplete replication and nuclease degradation and to extend the proliferation potential of germ and stem cells and most cancers. However, telomeres in somatic cells gradually shorten with age, ultimately leading to cellular senescence. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and diverse symptoms including bone marrow failure, immunodeficiency, and neurodevelopmental defects. HHS is caused by germline mutations in telomerase subunits, factors essential for its biogenesis and recruitment to telomeres, and in the helicase RTEL1. While diverse phenotypes were associated with RTEL1 deficiency, the telomeric role of RTEL1 affected in HHS is yet unknown. Inducible ectopic expression of wild-type RTEL1 in patient fibroblasts rescued the cells, enabled telomerase-dependent telomere elongation and suppressed the abnormal cellular phenotypes, while silencing its expression resulted in gradual telomere shortening. Our observations reveal an essential role of the RTEL1 C-terminus in facilitating telomerase action at the telomeric 3' overhang. Thus, the common etiology for HHS is the compromised telomerase action, resulting in telomere shortening and reduced lifespan of telomerase positive cells.

Identification of telomerase RNAs in species of the Yarrowia clade provides insights into the co-evolution of telomerase, telomeric repeats and telomere-binding proteins.

Telomeric repeats in fungi of the subphylum Saccharomycotina exhibit great inter- and intra-species variability in length and sequence. Such variations challenged telomeric DNA-binding proteins that co-evolved to maintain their functions at telomeres. Here, we compare the extent of co-variations in telomeric repeats, encoded in the telomerase RNAs (TERs), and the repeat-binding proteins from 13 species belonging to the Yarrowia clade. We identified putative TER loci, analyzed their sequence and secondary structure conservation, and predicted functional elements. Moreover, in vivo complementation assays with mutant TERs showed the functional importance of four novel TER substructures. The TER-derived telomeric repeat unit of all species, except for one, is 10 bp long and can be represented as 5'-TTNNNNAGGG-3', with repeat sequence variations occuring primarily outside the vertebrate telomeric motif 5'-TTAGGG-3'. All species possess a homologue of the Yarrowia lipolytica Tay1 protein, YlTay1p. In vitro, YlTay1p displays comparable DNA-binding affinity to all repeat variants, suggesting a conserved role among these species. Taken together, these results add significant insights into the co-evolution of TERs, telomeric repeats and telomere-binding proteins in yeasts.

Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection.

Universal minicircle sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind a single-stranded G-rich sequence, UMS, conserved at the replication origins of the mitochondrial (kinetoplast) DNA of trypanosomatids. Here, we report that Trypanosoma brucei TbUMSBP2, which has been previously proposed to function in the replication and segregation of the mitochondrial DNA, colocalizes with telomeres at the nucleus and is essential for their structure, protection and function. Knockdown of TbUMSBP2 resulted in telomere clustering in one or few foci, phosphorylation of histone H2A at the vicinity of the telomeres, impaired nuclear division, endoreduplication and cell growth arrest. Furthermore, TbUMSBP2 depletion caused rapid reduction in the G-rich telomeric overhang, and an increase in C-rich single-stranded telomeric DNA and in extrachromosomal telomeric circles. These results indicate that TbUMSBP2 is essential for the integrity and function of telomeres. The sequence similarity between the mitochondrial UMS and the telomeric overhang and the finding that UMSBPs bind both sequences suggest a common origin and/or function of these interactions in the replication and maintenance of the genomes in the two organelles. This feature could have converged or preserved during the evolution of the nuclear and mitochondrial genomes from their ancestral (likely circular) genome in early diverged protists.

Human RTEL1 stabilizes long G-overhangs allowing telomerase-dependent over-extension.

Telomere maintenance protects the cell against genome instability and senescence. Accelerated telomere attrition is a characteristic of premature aging syndromes including Dyskeratosis congenita (DC). Mutations in hRTEL1 are associated with a severe form of DC called Hoyeraal-Hreidarsson syndrome (HHS). HHS patients carry short telomeres and HHS cells display telomere damage. Here we investigated how hRTEL1 contributes to telomere maintenance in human primary as well as tumor cells. Transient depletion of hRTEL1 resulted in rapid telomere shortening only in the context of telomerase-positive cells with very long telomeres and high levels of telomerase. The effect of hRTEL1 on telomere length is telomerase dependent without impacting telomerase biogenesis or targeting of the enzyme to telomeres. Instead, RTEL1 depletion led to a decrease in both G-overhang content and POT1 association with telomeres with limited telomere uncapping. Strikingly, overexpression of POT1 restored telomere length but not the overhang, demonstrating that G-overhang loss is the primary defect caused by RTEL1 depletion. We propose that hRTEL1 contributes to the maintenance of long telomeres by preserving long G-overhangs, thereby facilitating POT1 binding and elongation by telomerase.

Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects.

The analysis of individuals with telomere defects may shed light on the delicate interplay of factors controlling genome stability, premature aging, and cancer. We herein describe two Coats plus patients with telomere and genomic defects; both harbor distinct, novel mutations in STN1, a member of the human CTC1-STN1-TEN1 (CST) complex, thus linking this gene for the first time to a human telomeropathy. We characterized the patients' phenotype, recapitulated it in a zebrafish model and rescued cellular and clinical aspects by the ectopic expression of wild-type STN1 or by thalidomide treatment. Interestingly, a significant lengthy control of the gastrointestinal bleeding in one of our patients was achieved by thalidomide treatment, exemplifying a successful bed-to-bench-and-back approach.

Unraveling the pathogenesis of Hoyeraal-Hreidarsson syndrome, a complex telomere biology disorder.

Hoyeraal-Hreidarsson (HH) syndrome is a multisystem genetic disorder characterized by very short telomeres and considered a clinically severe variant of dyskeratosis congenita. The main cause of mortality, usually in early childhood, is bone marrow failure. Mutations in several telomere biology genes have been reported to cause HH in about 60% of the HH patients, but the genetic defects in the rest of the patients are still unknown. Understanding the aetiology of HH and its diverse manifestations is challenging because of the complexity of telomere biology and the multiple telomeric and non-telomeric functions played by telomere-associated proteins in processes such as telomere replication, telomere protection, DNA damage response and ribosome and spliceosome assembly. Here we review the known clinical complications, molecular defects and germline mutations associated with HH, and elucidate possible mechanistic explanations and remaining questions in our understanding of the disease.

Inherited mutations in the helicase RTEL1 cause telomere dysfunction and Hoyeraal-Hreidarsson syndrome.

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.

Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo.

Telomerase is a ribonucleoprotein complex that extends the 3' ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C(+)•G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.

Identification of telomerase RNAs from filamentous fungi reveals conservation with vertebrates and yeasts.

Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5' splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.

The Trypanosoma brucei telomerase RNA (TER) homologue binds core proteins of the C/D snoRNA family.

Trypanosome protozoan parasites are the causative agents of devastating diseases. Trypanosome telomeres grow in an uncontrolled manner and the variant surface glycoprotein (VSG) genes are located in subtelomeric domains. The gene encoding telomerase reverse transcriptase (TERT) was identified and in this study, we describe the Trypanosoma brucei telomerase RNA (TER). TER RNA is bound by the core proteins of the C/D small nucleolar RNA (snoRNA) family and associates with the methyltransferase-associated protein (MTAP), whose homologue binds to mammalian TER. Silencing of TbTER resulted in telomere shortening. This is the first report of a TER that binds the C/D snoRNA core proteins.

"Poisoning" yeast telomeres distinguishes between redundant telomere capping pathways.

In most eukaryotes, telomeres are composed of tandem arrays of species-specific DNA repeats ending with a G-rich 3' overhang. In budding yeast, Cdc13 binds this overhang and recruits Ten1-Stn1 and the telomerase protein Est1 to protect (cap) and elongate the telomeres, respectively. To dissect and study the various pathways employed to cap and maintain the telomere end, we engineered telomerase to incorporate Tetrahymena telomeric repeats (G4T2) onto the telomeres of the budding yeast Kluyveromyces lactis. These heterologous repeats caused telomere-telomere fusions, cell cycle arrest at G2/M, and severely reduced viability--the hallmarks of telomere uncapping. Fusing Cdc13 or Est1 to universal minicircle sequence binding protein (UMSBP), a small protein that binds the single-stranded G4T2 repeats, rescued the cell viability and restored telomere capping, but not telomerase-mediated telomere maintenance. Surprisingly, Cdc13-UMSBP-mediated telomere capping was dependent on the homologous recombination factor Rad52, while Est1-UMSBP was not. Thus, our results distinguish between two, redundant, telomere capping pathways.