Structural conservation in the template/pseudoknot domain of vertebrate telomerase RNA from teleost fish to human

Abstract

Telomerase is an RNA-protein complex that includes a unique reverse transcriptase that catalyzes the addition of single-stranded telomere DNA repeats onto the 3′ ends of linear chromosomes using an integral telomerase RNA (TR) template. Vertebrate TR contains the template/pseudoknot (t/PK) and CR4/5 domains required for telomerase activity in vitro. All vertebrate pseudoknots include two subdomains: P2ab (helices P2a and P2b with a 5/6-nt internal loop) and the minimal pseudoknot (P2b-P3 and associated loops). A helical extension of P2a, P2a.1, is specific to mammalian TR. Using NMR, we investigated the structures of the full-length TR pseudoknot and isolated subdomains in Oryzias latipes (Japanese medaka fish), which has the smallest vertebrate TR identified to date. We determined the solution NMR structure and studied the dynamics of medaka P2ab, and identified all base pairs and tertiary interactions in the minimal pseudoknot. Despite differences in length and sequence, the structure of medaka P2ab is more similar to human P2ab than predicted, and the medaka minimal pseudoknot has the same tertiary interactions as the human pseudoknot. Significantly, although P2a.1 is not predicted to form in teleost fish, we find that it forms in the full-length pseudoknot via an unexpected hairpin. Model structures of the subdomains are combined to generate a model of t/PK. These results provide evidence that the architecture for the vertebrate t/PK is conserved from teleost fish to human. The organization of the t/PK on telomerase reverse transcriptase for medaka and human is modeled based on the cryoEM structure of Tetrahymena telomerase, providing insight into function.T elomeres are the physical ends of linear chromosomes, composed of telomeric DNA and associated proteins. After each round of cell replication, telomeres shorten because of incomplete 3′-end replication of telomere DNA repeats and nucleolytic processing. Telomerase, a specialized reverse transcriptase, is a large ribonucleoprotein that plays essential roles in protecting the integrity of linear chromosomes in most eukaryotic species (1). Most somatic cells do not contain detectable telomerase activity, and consequently, after 40-60 cell divisions, their telomeres shorten below a critical length [∼12.8 repeats (2)] that leads to replicative senescence or to apoptosis (3). Most cancer cell lines, on the other hand, have significant telomerase activity, which continuously replenishes their telomeres and allows them to divide indefinitely; this dichotomy has made telomerase inhibition an attractive target for the development of anticancer drugs (4, 5). Mutations in human telomerase proteins and RNA cause a number of inherited diseases, such as autosomal dominant dyskeratosis congenita, idiopathic pulmonary fibrosis, myelodysplasia, and aplastic anemia (6-10).The telomerase holoenzyme consists of a unique reverse transcriptase protein (telomerase reverse transcriptase; TERT), which contains the active site for nucleotide addition, an essential telomerase RNA (...

Progress in Human andTetrahymenaTelomerase Structure Determination

“…A direct correlation between pseudoknot stability and telomerase activity was demonstrated by thermodynamic analysis, through the use of nucleotide substitutions and compensatory mutations (157). Biochemical, mutational, and structural studies of yeast and medaka fish pseudoknots have revealed a conserved structure and confirmed the importance of the base triples for pseudoknot stability and telomerase function (Figure 3 a ) (17, 127, 147, 163, 171). A recent structure of the Tetrahymena pseudoknot revealed a more limited set of base triples, explaining in part its lower stability (Figure 3 a ) (18, 69).…”

NMR Studies of the Structure and Function of the HIV-1 5′-Leader

“…Although NMR spectroscopy has historically been used to determine structures of relatively small RNAs that typically comprise fewer than 60 nucleotides [67,68,69,70,71,72,73,74,75], the above studies have shown that structures can be probed by NMR spectroscopy in RNAs comprising up to 688 nucleotides. When used in combination with cryo-electron microscopy (cryoEM) or small-angle X-ray scattering (SAXS), 3D structural information can be obtained for relatively large protein:RNA complexes [68,69].…”

Structural biology of telomerase and its interaction at telomeres

“…A similar approach was recently used to model the t/PK of medaka fish ( Orzyis lapites ), which has the smallest vertebrate TER [46]. This structural study of medaka pseudoknot domains showed that the tertiary stem-loop interactions are identical to human TER except it lacks a single nt bulge near the stem-stem junction, the predicted 6 nt bulge-loop is actually a 5 nt loop with the same structure as in hTER, and the adjacent predicted single-strand region forms a small hairpin that stacks on the adjacent helix [18]. Based on the model structures of the medaka and human t/PK and the locations of TER elements in Tetrahymena , models of medaka and human TERT in complex with the t/PK were presented (Figure 2e).…”

“…The smFRET data of human TERT-TER complexes (discussed above) with primer bound (stalled) and actively synthesizing DNA showed three different positions of the pseudoknot fold relative to the template [47]. The authors propose that the pseudoknot moves during the telomere repeat synthesis cycle (pseudoknot tracking model); however, since they assumed a rigid structure for TERT in their Rosetta modeling they also suggest that the PK fold motion stabilizes the alternative conformation of TERT CTE proposed by Wang [18]. Most recently, a comprehensive mutational screen of TERT identified a ssDNA retention surface (SRS) within the CTE (Figure 3c), which is proposed to maintain placement of the 3′ end of the nascent DNA telomere repeat in the active site during template translocation, thereby contributing to repeat addition processivity [56].…”

“…Somewhat more rapid structural progress had been made with telomere binding proteins, especially from yeasts [12]. During the past couple of years, a surge of progress has been made on structural biology of telomerase, including reports of the first cryo-electron microscopy (cryo-EM) structure of telomerase, from Tetrahymena [13], crystal structures of TERT and TERT-interacting protein domains [14–17], telomerase RNA structures and models [18,19], and identification in Tetrahymena telomerase holoenzyme of human homologues of telomere-associated proteins [13,20] that have provided a more unified view of telomerase interaction at telomeres [21]. Here we review these and other studies published since 2015 on structural investigations of primarily Tetrahymena and vertebrate telomerase and its interaction at telomeres.…”