Analysis of Telomerase RNA Structure in Physcomitrium patens Indicates Functionally Relevant Transitions Between OPEN and CLOSED Conformations.

Telomerase RNA (TR) conformation determines its function as a template for telomere synthesis and as a scaffold for the assembly of the telomerase nucleoprotein complex. Experimental analyses of TR secondary structure using DMS-Map Seq and SHAPE-Map Seq techniques show its CLOSED conformation as the consensus structure where the template region cannot perform its function. Our data show that the apparent discrepancy between experimental results and predicted TR functional conformation, mostly ignored in published studies, can be explained using data analysis based on single-molecule structure prediction from individual sequencing reads by the recently established DaVinci method. This method results in several clusters of secondary structures reflecting the structural dynamics of TR, possibly related to its multiple functional states. Interestingly, the presumed active (OPEN) conformation of TR corresponds to a minor fraction of TR under in vivo conditions. Therefore, structural polymorphism and dynamic TR transitions between CLOSED and OPEN conformations may be involved in telomerase activity regulation as a switch that functions independently of total TR transcript levels.

Biochemical and structural characterization of the RT domain of Leishmania sp. telomerase reverse transcriptase.

BACKGROUND: The Leishmania genus comprises parasites that cause leishmaniasis, a neglected disease spread worldwide. Leishmania sp. telomeres are composed of TTAGGG repeats maintained by telomerase. In most eukaryotes, the enzyme minimal complex contains the TER (telomerase RNA) and the TERT (telomerase reverse transcriptase) components. The TERT holds the enzyme catalytic core and is formed by four structural and functional domains (TEN, Telomerase Essential N-terminal; TRBD, Telomerase RNA Binding Domain; RT, the reverse transcriptase domain and CTE, C-Terminal Extension domain). METHODS AND RESULTS: Amino acid sequence alignments, protein structure prediction analysis, and protein: nucleic acid interaction assays were used to show that the Leishmania major RT domain preserves the canonical structural elements found in higher eukaryotes, including the canonical motifs and the aspartic acid residues that stabilize the Mg2+ ion cofactor. Furthermore, amino acid substitutions specific to the Leishmania genus and partial conservation of the residues involved with nucleic acid interactions are shown. The purified recombinant Leishmania RT protein is biochemically active and interacts with the G-rich telomeric strand and the TER template sequence. CONCLUSION: Our results highlight that the telomerase catalysis mechanism is conserved in a pathogen of medical importance despite the structural peculiarities present in the parasite's RT domain.

Real-time detection of human telomerase DNA synthesis by multiplexed single-molecule FRET.

Genomic stability in proliferating cells critically depends on telomere maintenance by telomerase reverse transcriptase. Here we report the development and proof-of-concept results of a single-molecule approach to monitor the catalytic activity of human telomerase in real time and with single-nucleotide resolution. Using zero-mode waveguides and multicolor FRET, we recorded the processive addition of multiple telomeric repeats to individual DNA primers. Unlike existing biophysical and biochemical tools, the novel approach enables the quantification of nucleotide-binding kinetics before nucleotide incorporation. Moreover, it provides a means to dissect the unique translocation dynamics that telomerase must undergo after synthesis of each hexameric DNA repeat. We observed an unexpectedly prolonged binding dwell time of dGTP in the enzyme active site at the start of each repeat synthesis cycle, suggesting that telomerase translocation is composed of multiple rate-contributing sub-steps that evade classical biochemical analysis.

Orchestrating nucleic acid-protein interactions at chromosome ends: telomerase mechanisms come into focus.

Telomerase is a special reverse transcriptase ribonucleoprotein dedicated to the synthesis of telomere repeats that protect chromosome ends. Among reverse transcriptases, telomerase is unique in using a stably associated RNA with an embedded template to synthesize a specified sequence. Moreover, it is capable of iteratively copying the same template region (repeat addition processivity) through multiple rounds of RNA-DNA unpairing and reannealing, that is, the translocation reaction. Biochemical analyses of telomerase over the past 3 decades in protozoa, fungi and mammals have identified structural elements that underpin telomerase mechanisms and have led to models that account for the special attributes of telomerase. Notably, these findings and models can now be interpreted and adjudicated through recent cryo-EM structures of Tetrahymena and human telomerase holoenzyme complexes in association with substrates and regulatory proteins. Collectively, these structures reveal the intricate protein-nucleic acid interactions that potentiate telomerase's unique translocation reaction and clarify how this enzyme reconfigures the basic reverse transcriptase scaffold to craft a polymerase dedicated to the synthesis of telomere DNA. Among the many new insights is the resolution of the telomerase 'anchor site' proposed more than 3 decades ago. The structures also highlight the nearly universal conservation of a protein-protein interface between an oligonucleotide/oligosaccharide-binding (OB)-fold regulatory protein and the telomerase catalytic subunit, which enables spatial and temporal regulation of telomerase function in vivo. In this Review, we discuss key features of the structures in combination with relevant functional analyses. We also examine conserved and divergent aspects of telomerase mechanisms as gleaned from studies in different model organisms.

Structure of LARP7 Protein p65-telomerase RNA Complex in Telomerase Revealed by Cryo-EM and NMR.

La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3'-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains-N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65-TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65-TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3' terminal U nucleotides, while LaM and αN additionally interact with TER pseudoknot, and LaM with stem 1 and 5' end. Our results reveal the extensive p65-TER interactions that promote TER 3'-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components.

Probing the general base for DNA polymerization in telomerase: a molecular dynamics investigation.

Telomerase is an RNA-dependent DNA polymerase that plays a role in the maintenance of the 3' end of the eukaryotic chromosome, known as a telomere, by catalyzing the DNA polymerization reaction in cancer and embryonic stem cells. The detailed molecular details of the DNA polymerization by telomerase, especially the general base for deprotonating the terminal 3'-hydroxyl, which triggers the chemical reaction, remain elusive. We conducted a computational investigation using hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations to probe the detailed mechanism of the reaction. Our simulations started with the telomerase:RNA:DNA:dNTP ternary complex, and by using enhanced sampling QM/MM MD simulations, we probed the general base involved directly in the polymerization. We report the participation of an aspartate (Asp344) coordinated to Mg and an active site water molecule, jointly acting as a base during nucleic acid addition. The Asp344 residue remains transiently protonated during the course of the reaction, and later it deprotonates by transferring its proton to the water at the end of the reaction.

Complex interaction network revealed by mutation of human telomerase 'insertion in fingers' and essential N-terminal domains and the telomere protein TPP1.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here, we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres, and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

Telomeric chromatin structure.

Eukaryotic DNA is packaged into nucleosomes, which further condenses into chromosomes. The telomeres, which form the protective end-capping of chromosomes, play a pivotal role in ageing and cancer. Recently, significant advances have been made in understanding the nucleosomal and telomeric chromatin structure at the molecular level. In addition, recent studies shed light on the nucleosomal organisation at telomeres revealing its ultrastructural organisation, the atomic structure at the nucleosome level, its dynamic properties, and higher-order packaging of telomeric chromatin. Considerable advances have furthermore been made in understanding the structure, function and organisation of shelterin, telomerase and CST complexes. Here we discuss these recent advances in the organisation of telomeric nucleosomes and chromatin and highlight progress in the structural understanding of shelterin, telomerase and CST complexes.

Telomerase structural biology comes of age.

Telomerase is an RNA-protein complex comprising telomerase reverse transcriptase, a non-coding telomerase RNA, and proteins involved in biogenesis, assembly, localization, or recruitment. Telomerase synthesizes the telomeric DNA at the 3'-ends of linear chromosomes. During the past decade, structural studies have defined the architecture of Tetrahymena and human telomerase as well as protein and RNA domain structures, but high-resolution details of interactions remained largely elusive. In the past two years, several sub-4 Å cryo-electron microscopy structures of telomerase were published, including Tetrahymena telomerase at different steps of telomere repeat addition and human telomerase with telomere shelterin proteins that recruit telomerase to telomeres. These and other recent structural studies have expanded our understanding of telomerase assembly, mechanism, recruitment, and mutations leading to disease.

Interface of G-quadruplex with both stabilizing and destabilizing ligands for targeting various diseases.

Guanine-rich DNA sequences may fold back into non-canonical four-stranded secondary structures termed as G-quadruplexes. The role of G-quadruplexes has already been well established in different diseases like cancer, neurological and viral disorders etc. Also, several small molecules have been reported, which can influence the involvement of G-quadruplexes either through stabilization or destabilization in the cellular environment. Growing statistics have associated G-quadruplex assemblies to a discrete biological process in vivo, including DNA replication, transcription, genomic stability, and epigenetic regulation. DNA G-quadruplex existence in human telomere is well recognized attractive target for anticancer drugs. G-quadruplex-interactive ligands have been known to prevent telomerase access as well as telomere capping. To the best of our understanding, the role of G-quadruplexes in virology, neuropharmacology, cancer progression and its treatment has not been discussed on a single platform till date. This review aims to enhance our knowledge regarding these magical sticky quadruplex structures, which have been quite significantly proved to be the part of many cellular processes along with their established in vivo existence. Understanding regarding stabilizing or destabilizing ligands of these multistranded guanine quadruplex structures might be proved as the facilitator of drug discovery process for many incurable diseases in future.

Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.

Structural basis of human telomerase recruitment by TPP1-POT1.

Telomerase maintains genome stability by extending the 3' telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. We report structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Our structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Our findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.

Telomerase ribonucleoprotein and genome integrity-An emerging connection in protozoan parasites.

Telomerase has an established role in telomere maintenance in eukaryotes. However, recent studies have begun to implicate telomerase in cellular roles beyond telomere maintenance. Specifically, evidence is emerging of cross-talks between telomerase mediated telomere homeostasis and DNA repair pathways. Telomere shortening due to the end replication problem is a constant threat to genome integrity in eukaryotic cells. This poses a particular problem in unicellular parasitic protists because their major virulence genes are located at the subtelomeric loci. Although telomerase is the major regulator of telomere lengthening in eukaryotes, it is less studied in the ancient eukaryotes, including clinically important human pathogens. Recent research is highlighting interplay between telomerase and the DNA damage response in human parasites. The importance of this interplay in pathogen virulence is only beginning to be illuminated, including the potential to highlight novel developmental regulation of telomerase in parasites who transition between multiple developmental stages throughout their life cycle. In this review, we will discuss the telomerase ribonucleoprotein enzyme and DNA repair pathways with emerging views in human parasites to give a broader perspective of the possible connection of telomere, telomerase, and DNA repair pathways across eukaryotic lineages and highlight their potential role in pathogen virulence. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Stem cells at odds with telomere maintenance and protection.

Telomeres are distinctive structures that protect the ends of linear chromosomes and ensure genome stability. They are composed of long tracks of repetitive and G-rich DNA that is bound by shelterin, a dedicated six-subunit protein complex. In somatic cells, shelterin protects telomeres from the DNA damage response and regulates telomere length. Telomere repeats are replenished by telomerase, a specialized ribonucleoprotein composed of telomerase reverse transcriptase and an integral RNA component. Telomere protection and telomerase regulation have been primarily studied in somatic cells. However, recent evidence points out striking differences in the context of embryonic stem cells (ESCs). In this review, we discuss insights into telomere protection in ESCs versus somatic cells and summarize findings on telomerase regulation as a function of pluripotency.

In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei.

Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5'-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT-/- cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development.

The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses.

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3' end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase.

Structural biology of human telomerase: progress and prospects.

Telomerase ribonucleoprotein was discovered over three decades ago as a specialized reverse transcriptase that adds telomeric repeats to the ends of linear eukaryotic chromosomes. Telomerase plays key roles in maintaining genome stability; and its dysfunction and misregulation have been linked to different types of cancers and a spectrum of human genetic disorders. Over the years, a wealth of genetic and biochemical studies of human telomerase have illuminated its numerous fascinating features. Yet, structural studies of human telomerase have lagged behind due to various challenges. Recent technical developments in cryo-electron microscopy have allowed for the first detailed visualization of the human telomerase holoenzyme, revealing unprecedented insights into its active site and assembly. This review summarizes the cumulative work leading to the recent structural advances, as well as highlights how the future structural work will further advance our understanding of this enzyme.

CST does not evict elongating telomerase but prevents initiation by ssDNA binding.

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

Evolution of plant telomerase RNAs: farther to the past, deeper to the roots.

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.