Structural basis of template-boundary definition in Tetrahymena telomerase

Jansson, Linnea I.; Akiyama, Ben M; Ooms, Alexandra N.; Lü, Cheng; Rubin, Seth M.; Stone, Michael D.

doi:10.1038/nsmb.3101

Cited by 47 publications

(77 citation statements)

References 32 publications

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“…The P1.1 stem-loop resembles the template-adjacent helix in fungal TRs and Helix II in ciliate TRs (Podlevsky and Chen 2012). Fungal and ciliate template-adjacent helices function similarly by physically restricting the residues in the template-flanking region from being used as the template (Tzfati et al 2000;Jansson et al 2015). Instead of using a template-adjacent helix, vertebrate TR template boundary is defined by the more distant P1b helix and a single-stranded linker with a specific length to the template (Chen and Greider 2003).…”

Section: Discussionmentioning

confidence: 99%

“…2). However, it remains unclear whether the echinoderm P1.1 stem-loop functions as a TERT protein binding site, similar to ciliate TR helix II, whereby TERT protein binding impedes the use of nucleotides as the template from outside the template boundary (Autexier and Greider 1995;Lai et al 2002;Jansson et al 2015). Moreover, as demonstrated in this study, the echinoderm P1.1 helix can be entirely removed and replaced with a specific linker length to the P1b helix which functions as the new template boundary element, using a mechanism identical to that of vertebrate TRs (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Structure and function of echinoderm telomerase RNA

Podlevsky

Chen

2015

RNA

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Telomerase is a ribonucleoprotein (RNP) enzyme that requires an integral telomerase RNA (TR) subunit, in addition to the catalytic telomerase reverse transcriptase (TERT), for enzymatic function. The secondary structures of TRs from the three major groups of species, ciliates, fungi, and vertebrates, have been studied extensively and demonstrate dramatic diversity. Herein, we report the first comprehensive secondary structure of TR from echinoderms-marine invertebrates closely related to vertebratesdetermined by phylogenetic comparative analysis of 16 TR sequences from three separate echinoderm classes. Similar to vertebrate TR, echinoderm TR contains the highly conserved template/pseudoknot and H/ACA domains. However, echinoderm TR lacks the ancestral CR4/5 structural domain found throughout vertebrate and fungal TRs. Instead, echinoderm TR contains a distinct simple helical region, termed eCR4/5, that is functionally equivalent to the CR4/5 domain. The urchin and brittle star eCR4/5 domains bind specifically to their respective TERT proteins and stimulate telomerase activity. Distinct from vertebrate telomerase, the echinoderm TR template/pseudoknot domain with the TERT protein is sufficient to reconstitute significant telomerase activity. This gain-of-function of the echinoderm template/pseudoknot domain for conferring telomerase activity presumably facilitated the rapid structural evolution of the eCR4/5 domain throughout the echinoderm lineage. Additionally, echinoderm TR utilizes the template-adjacent P1.1 helix as a physical template boundary element to prevent nontelomeric DNA synthesis, a mechanism used by ciliate and fungal TRs. Thus, the chimeric and eccentric structural features of echinoderm TR provide unparalleled insights into the rapid evolution of telomerase RNP structure and function.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structure and function of echinoderm telomerase RNA

Podlevsky

Chen

2015

RNA

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“…The TBE is a stem loop located only a few nucleotides upstream of the RNA template and is coordinated by the T, CP, and CP2 motifs in Tetrahymena thermophila (13,14) or T, CP, and TFLY in vertebrates (8). These TBE-binding motifs are located at the interface of the TRBD and fingers domains, and together they form a well defined indentation on the surface of the protein providing the platform for a network of nonspecific interactions with the TBE (13).…”

Section: Edited By Joel Gottesfeldmentioning

confidence: 99%

“…Binding of the TBE to TRBD positions the RNA template at the active site of the enzyme thus promoting nucleotide binding and selectivity (3). We previously proposed, and it was subsequently shown, that the TRBD-TBE interaction provides the steric block that prevents TERT from replicating beyond the 5Ј end of the RNA template thus promoting telomerase repeat addition processivity (2,4,13).…”

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confidence: 99%

Structural Analysis Reveals the Deleterious Effects of Telomerase Mutations in Bone Marrow Failure Syndromes

Hoffman

Rice

Skordalakes

2017

Journal of Biological Chemistry

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Edited by Joel GottesfeldNaturally occurring mutations in the ribonucleoprotein reverse transcriptase, telomerase, are associated with the bone marrow failure syndromes dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis. However, the mechanism by which these mutations impact telomerase function remains unknown. Here we present the structure of the human telomerase C-terminal extension (or thumb domain) determined by the method of single-wavelength anomalous diffraction to 2.31 Å resolution. We also used direct telomerase activity and nucleic acid binding assays to explain how naturally occurring mutations within this portion of telomerase contribute to human disease. The single mutations localize within three highly conserved regions of the telomerase thumb domain referred to as motifs E-I (thumb loop and helix), E-II, and E-III (the FVYL pocket, comprising the hydrophobic residues Phe-1012, Val-1025, Tyr-1089, and Leu-1092). Biochemical data show that the mutations associated with dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis disrupt the binding between the protein subunit reverse transcriptase of the telomerase and its nucleic acid substrates leading to loss of telomerase activity and processivity. Collectively our data show that although these mutations do not alter the overall stability or expression of telomerase reverse transcriptase, these rare genetic disorders are associated with an impaired telomerase holoenzyme that is unable to correctly assemble with its nucleic acid substrates, leading to incomplete telomere extension and telomere attrition, which are hallmarks of these diseases.Human telomerase is a ribonucleoprotein reverse transcriptase that replicates the ends of eukaryotic chromosomes (1). The protein subunit, TERT, 2 consists of several domains (TEN, TRBD, RT (fingers and palm), and thumb) organized into a closed, ring configuration, generating a large cavity in the interior of the ring and where the RNA template and the DNA bind during telomere elongation (2-5). The closed configuration of the TERT ring is stabilized by extensive interactions between the thumb and TRBD domains as well as by protein-RNA interactions (3, 6). Several invariable motifs in the interior cavity of the TERT ring coordinate the RNA template and telomeric overhang (RNADNA hybrid) and position the 3Ј end of the DNA for catalysis (2, 3). These include motifs E-I and E-II of the thumb domain, which bind the RNADNA hybrid and stabilize the telomerase elongation complex (2, 7); the primer grip region that guides the DNA at the active site of the enzyme and motifs 2 and BЈ of the fingers and palm domain, respectively, which position the RNA template above the active site of the enzyme for nucleotide binding and selectivity (2, 3).Current evidence shows that the RNA binding domain of telomerase (TRBD) binds the template boundary element (TBE) and the activation domain (CR4/5) of telomerase RNA (8 -12). The TBE is a stem loop located only a few nucleotides upstream of the R...

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“…9 C and D). In Tetrahymena, the bottom of S2 and the flanking singlestranded residues of the TBE wrap on either side of the TRBD, serving as an anchor to prevent the use of nontemplate nucleotides for replication (47). In the medaka model, the bottom of P1 and flanking single strands of the TBE are positioned similarly.…”

Section: Resultsmentioning

confidence: 99%

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

Wang

Yesselman

Zhang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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 (...

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Structural basis of template-boundary definition in Tetrahymena telomerase

Cited by 47 publications

References 32 publications

Structure and function of echinoderm telomerase RNA

Structure and function of echinoderm telomerase RNA

Structural Analysis Reveals the Deleterious Effects of Telomerase Mutations in Bone Marrow Failure Syndromes

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

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