Joshua D. Podlevsky

The Telomerase Database

Telomerase is a ribonucleoprotein enzyme that extends DNA at the chromosome ends in most eukaryotes. Since 1985, telomerase has been studied intensively and components of the telomerase complex have been identified from over 160 eukaryotic species. In the last two decades, there has been a growing interest in studying telomerase owing to its vital role in chromosome stability and cellular immortality. To keep up with the remarkable explosion of knowledge about telomerase, we compiled information related to telomerase in an exhaustive database called the Telomerase Database (http://telomerase.asu.edu/). The Telomerase Database provides comprehensive information about (i) sequences of the RNA and protein subunits of telomerase, (ii) sequence alignments based on the phylogenetic relationship and structure, (iii) secondary structures of the RNA component and tertiary structures of various subunits of telomerase, (iv) mutations of telomerase components found in human patients and (v) active researchers who contributed to the wealth of current knowledge on telomerase. The information is hierarchically organized by the components, i.e. the telomerase reverse transcriptase (TERT), telomerase RNA (TR) and other telomerase-associated proteins. The Telomerase Database is a useful resource especially for researchers who are interested in investigating the structure, function, evolution and medical relevance of the telomerase enzyme.

It all comes together at the ends: Telomerase structure, function, and biogenesis

Telomerase is a reverse transcriptase specialized in the addition of telomeric DNA repeats onto the ends of chromosomes. Telomere extension offsets the loss of telomeric repeats from the failure of DNA polymerases to fully replicate linear chromosome ends. Telomerase functions as a ribonucleoprotein, requiring an integral telomerase RNA (TR) component, in addition to the catalytic telomerase reverse transcriptase (TERT). Extensive studies have identified numerous structural and functional features within the TR and TERT essential for activity. A number of accessory proteins have also been identified with various functions in enzyme biogenesis, localization, and regulation. Understanding the molecular mechanism of telomerase function has significance for the development of therapies for telomere-mediated disorders and cancer. Here we review telomerase structural and functional features, and the techniques for assessing telomerase dysfunction.

Telomerase mutations in smokers with severe emphysema

Mutations in the essential telomerase genes TERT and TR cause familial pulmonary fibrosis; however, in telomerase-null mice, short telomeres predispose to emphysema after chronic cigarette smoke exposure. Here, we tested whether telomerase mutations are a risk factor for human emphysema by examining their frequency in smokers with chronic obstructive pulmonary disease (COPD). Across two independent cohorts, we found 3 of 292 severe COPD cases carried deleterious mutations in TERT (1%). This prevalence is comparable to the frequency of alpha-1 antitrypsin deficiency documented in this population. The TERT mutations compromised telomerase catalytic activity, and mutation carriers had short telomeres. Telomerase mutation carriers with emphysema were predominantly female and had an increased incidence of pneumothorax. In families, emphysema showed an autosomal dominant inheritance pattern, along with pulmonary fibrosis and other telomere syndrome features, but manifested only in smokers. Our findings identify germline mutations in telomerase as a Mendelian risk factor for COPD susceptibility that clusters in autosomal dominant families with telomere-mediated disease including pulmonary fibrosis.

A novel motif in telomerase reverse transcriptase regulates telomere repeat addition rate and processivity

Telomerase is a specialized reverse transcriptase that adds telomeric DNA repeats onto chromosome termini. Here, we characterize a new telomerase-specific motif, called motif 3, in the catalytic domain of telomerase reverse transcriptase, that is crucial for telomerase function and evolutionally conserved between vertebrates and ciliates. Comprehensive mutagenesis of motif 3 identified mutations that remarkably increase the rate or alter the processivity of telomere repeat addition. Notably, the rate and processivity of repeat addition are affected independently by separate motif 3 mutations. The processive telomerase action relies upon a template translocation mechanism whereby the RNA template and the telomeric DNA strand separate and realign between each repeat synthesis. By analyzing the mutant telomerases reconstituted in vitro and in cells, we show that the hyperactive mutants exhibit higher repeat addition rates and faster enzyme turnovers, suggesting higher rates of strand-separation during template translocation. In addition, the strong correlation between the processivity of the motif 3 mutants and their ability to use an 8 nt DNA primer, suggests that motif 3 facilitates realignment between the telomeric DNA and the template RNA following strand-separation. These findings support motif 3 as a key determinant for telomerase activity and processivity.

Loss-of-function mutations in the RNA biogenesis factor NAF1 predispose to pulmonary fibrosis–emphysema

Mutations in the RNA biogenesis factor NAF1 cause short telomeres and, consequently, age-related lung disease in people. Tidy telomeres make for healthier lungs Telomeres are the protective caps that prevent the ends of chromosomes from unraveling. People carrying mutations in the protein or RNA component of telomerase, the enzyme that makes telomeres have short telomeres and a serious and often fatal lung disease—pulmonary fibrosis. Now, Stanley et al. find in several patients that other mutations, specifically those that interfere with RNA biogenesis, can also cause both short telomeres and lung disease. This work expands our understanding of how telomeres are maintained and their role in human disease. Chronic obstructive pulmonary disease and pulmonary fibrosis have been hypothesized to represent premature aging phenotypes. At times, they cluster in families, but the genetic basis is not understood. We identified rare, frameshift mutations in the gene for nuclear assembly factor 1, NAF1, a box H/ACA RNA biogenesis factor, in pulmonary fibrosis–emphysema patients. The mutations segregated with short telomere length, low telomerase RNA levels, and extrapulmonary manifestations including myelodysplastic syndrome and liver disease. A truncated NAF1 was detected in cells derived from patients, and, in cells in which the frameshift mutation was introduced by genome editing, telomerase RNA levels were reduced. The mutant NAF1 lacked a conserved carboxyl-terminal motif, which we show is required for nuclear localization. To understand the disease mechanism, we used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) to generate Naf1+/− mice and found that they had half the levels of telomerase RNA. Other box H/ACA RNA levels were also decreased, but rRNA pseudouridylation, which is guided by snoRNAs, was intact. Moreover, first-generation Naf1+/− mice showed no evidence of ribosomal pathology. Our data indicate that disease in NAF1 mutation carriers is telomere-mediated; they show that NAF1 haploinsufficiency selectively disturbs telomere length homeostasis by decreasing the levels of telomerase RNA while sparing rRNA pseudouridylation.

RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

Telomerase synthesizes telomeric DNA repeats onto chromosome termini from an intrinsic RNA template. The processive synthesis of DNA repeats relies on a unique, yet poorly understood, mechanism whereby the telomerase RNA template translocates and realigns with the DNA primer after synthesizing each repeat. Here, we provide evidence that binding of the realigned RNA/DNA hybrid by the active site is an essential step for template translocation. Employing a template‐free human telomerase system, we demonstrate that the telomerase active site directly binds to RNA/DNA hybrid substrates for DNA polymerization. In telomerase processivity mutants, the template‐translocation efficiency correlates with the affinity for the RNA/DNA hybrid substrate. Furthermore, the active site is unoccupied during template translocation as a 5 bp extrinsic RNA/DNA hybrid effectively reduces the processivity of the template‐containing telomerase. This suggests that strand separation and template realignment occur outside the active site, preceding the binding of realigned hybrid to the active site. Our results provide new insights into the ancient RNA/DNA hybrid binding ability of telomerase and its role in template translocation.

The common ancestral core of vertebrate and fungal telomerase RNAs

Telomerase is a ribonucleoprotein with an intrinsic telomerase RNA (TER) component. Within yeasts, TER is remarkably large and presents little similarity in secondary structure to vertebrate or ciliate TERs. To better understand the evolution of fungal telomerase, we identified 74 TERs from Pezizomycotina and Taphrinomycotina subphyla, sister clades to budding yeasts. We initially identified TER from Neurospora crassa using a novel deep-sequencing–based approach, and homologous TER sequences from available fungal genome databases by computational searches. Remarkably, TERs from these non-yeast fungi have many attributes in common with vertebrate TERs. Comparative phylogenetic analysis of highly conserved regions within Pezizomycotina TERs revealed two core domains nearly identical in secondary structure to the pseudoknot and CR4/5 within vertebrate TERs. We then analyzed N. crassa and Schizosaccharomyces pombe telomerase reconstituted in vitro, and showed that the two RNA core domains in both systems can reconstitute activity in trans as two separate RNA fragments. Furthermore, the primer-extension pulse-chase analysis affirmed that the reconstituted N. crassa telomerase synthesizes TTAGGG repeats with high processivity, a common attribute of vertebrate telomerase. Overall, this study reveals the common ancestral cores of vertebrate and fungal TERs, and provides insights into the molecular evolution of fungal TER structure and function.

A self-regulating template in human telomerase

Significance The telomerase enzyme is essential for maintaining the replicative capacity of highly prolific cells, such as stem cells and cancer, by synthesizing telomeric DNA onto chromosome ends. Telomerase functions as an RNA–protein complex with an integral telomerase RNA (TR) component. While the templates from all other reverse transcriptases (RTs) merely specify the sequence for nucleotide addition, we found that the human TR template is embedded with a single-residue pausing signal for regulating DNA synthesis. Mutation of this pausing signal alters the fundamental function of telomerase for synthesizing exact telomeric DNA repeats. This is the first instance, to our knowledge, of a single-residue pausing signal found in the RNA template of an RT. Telomerase is a specialized reverse transcriptase (RT) containing an intrinsic telomerase RNA (TR) component. It synthesizes telomeric DNA repeats, (GGTTAG)n in humans, by reiteratively copying a precisely defined, short template sequence from the integral TR. The specific mechanism of how the telomerase active site uses this short template region accurately and efficiently during processive DNA repeat synthesis has remained elusive. Here we report that the human TR template, in addition to specifying the DNA sequence, is embedded with a single-nucleotide signal to pause DNA synthesis. After the addition of a dT residue to the DNA primer, which is specified by the 49 rA residue in the template, telomerase extends the DNA primer with three additional nucleotides and then pauses DNA synthesis. This sequence-defined pause site coincides precisely with the helix paired region 1 (P1)-defined physical template boundary and precludes the incorporation of nontelomeric nucleotides from residues outside the template region. Furthermore, this sequence-defined pausing mechanism is a key determinant, in addition to the P1-defined template boundary, for generating the characteristic 6-nt ladder banding pattern of telomeric DNA products in vitro. In the absence of the pausing signal, telomerase stalls nucleotide addition at multiple sites along the template, generating DNA products with heterogeneous terminal repeat registers. Our findings demonstrate that this unique self-regulating mechanism of the human TR template is essential for high-fidelity synthesis of DNA repeats.

Evolutionary perspectives of telomerase RNA structure and function

ABSTRACT Telomerase is the eukaryotic solution to the ‘end-replication problem’ of linear chromosomes by synthesising the highly repetitive DNA constituent of telomeres, the nucleoprotein cap that protects chromosome termini. Functioning as a ribonucleoprotein (RNP) enzyme, telomerase is minimally composed of the highly conserved catalytic telomerase reverse transcriptase (TERT) and essential telomerase RNA (TR) component. Beyond merely providing the template for telomeric DNA synthesis, TR is an innate telomerase component and directly facilitates enzymatic function. TR accomplishes this by having evolved structural elements for stable assembly with the TERT protein and the regulation of the telomerase catalytic cycle. Despite its prominence and prevalence, TR has profoundly diverged in length, sequence, and biogenesis pathway among distinct evolutionary lineages. This diversity has generated numerous structural and mechanistic solutions for ensuring proper RNP formation and high fidelity telomeric DNA synthesis. Telomerase provides unique insights into RNA and protein coevolution within RNP enzymes.

Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach

Telomerase is a ribonucleoprotein (RNP) enzyme essential for telomere maintenance and chromosome stability. While the catalytic telomerase reverse transcriptase (TERT) protein is well conserved across eukaryotes, telomerase RNA (TR) is extensively divergent in size, sequence, and structure. This diversity prohibits TR identification from many important organisms. Here we report a novel approach for TR discovery that combines in vitro TR enrichment from total RNA, next-generation sequencing, and a computational screening pipeline. With this approach, we have successfully identified TR from Strongylocentrotus purpuratus (purple sea urchin) from the phylum Echinodermata. Reconstitution of activity in vitro confirmed that this RNA is an integral component of sea urchin telomerase. Comparative phylogenetic analysis against vertebrate TR sequences revealed that the purple sea urchin TR contains vertebrate-like template-pseudoknot and H/ACA domains. While lacking a vertebrate-like CR4/5 domain, sea urchin TR has a unique central domain critical for telomerase activity. This is the first TR identified from the previously unexplored invertebrate clade and provides the first glimpse of TR evolution in the deuterostome lineage. Moreover, our TR discovery approach is a significant step toward the comprehensive understanding of telomerase RNP evolution.