Structural basis for protein-RNA recognition in telomerase

Huang, Jing; Brown, Andrew F.; Wu, Jian; Xue, Jing; Bley, Christopher J.; Rand, Dustin P.; Wu, Lijie; Zhang, Rongguang; Chen, Julian J.‐L.; Lei, Ming

doi:10.1038/nsmb.2819

Cited by 73 publications

(146 citation statements)

References 38 publications

Supporting

Mentioning

142

Contrasting

Order By: Relevance

“…3A and B) (18,19). Within the CR4/5 domain, the loop and base of helix P6.1 have been shown to be especially important (40)(41)(42). Using our circular-permutation analysis, we found that disrupting the backbone between CR4/5 and the core is tolerated to different degrees.…”

Section: Resultsmentioning

confidence: 97%

“…These mutations could affect the essential CR4/5 TERT-binding function (18) or RNA conformational changes. For instance, the medaka fish CR4/5 binds to the RNA-binding domain of TERT and undergoes a dramatic rearrangement of the three-way junction compared to unbound RNA (40). In contrast, circular permutations at the 5= and 3= borders of CR4/5 (CP242 and CP328) show strong or detectable activity (Fig.…”

Section: (6)mentioning

confidence: 99%

See 1 more Smart Citation

Physical Connectivity Mapping by Circular Permutation of Human Telomerase RNA Reveals New Regions Critical for Activity and Processivity

Mefford

Zappulla

2016

Molecular and Cellular Biology

View full text Add to dashboard Cite

Telomerase is a specialized ribonucleoprotein complex that extends the 3= ends of chromosomes to counteract telomere shortening. However, increased telomerase activity is associated with ϳ90% of human cancers. The telomerase enzyme minimally requires an RNA (hTR) and a specialized reverse transcriptase protein (TERT) for activity in vitro. Understanding the structurefunction relationships within hTR has important implications for human disease. For the first time, we have tested the physicalconnectivity requirements in the 451-nucleotide hTR RNA using circular permutations, which reposition the 5= and 3= ends. Our extensive in vitro analysis identified three classes of hTR circular permutants with altered function. First, circularly permuting 3= of the template causes specific defects in repeat-addition processivity, revealing that the template recognition element found in ciliates is conserved in human telomerase RNA. Second, seven circular permutations residing within the catalytically important core and CR4/5 domains completely abolish telomerase activity, unveiling mechanistically critical portions of these domains. Third, several circular permutations between the core and CR4/5 significantly increase telomerase activity. Our extensive circular permutation results provide insights into the architecture and coordination of human telomerase RNA and highlight where the RNA could be targeted for the development of antiaging and anticancer therapeutics. Linear eukaryotic chromosomes terminate in repeated DNA sequences, called telomeres, which are bound by specific proteins to protect the ends from degradation and detrimental end joining. However, these termini present an end-replication problem that most eukaryotes overcome by utilizing the ribonucleoprotein (RNP) complex telomerase. Telomerase comprises an RNA (hTR in humans) and a reverse transcriptase (TERT), which catalyzes telomere addition. It has been shown that telomeres shorten with aging, and telomerase upregulation occurs in ϳ90% of human cancers (1). Furthermore, mutations in telomerase components have been linked to a variety of short-telomere syndromes, such as dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia (2). Thus, understanding the structure-function relationships of human telomerase RNA is crucial to combat a range of human ailments.During the telomerase catalytic cycle, a short region of the telomerase RNA, known as the template, pairs with the laggingstrand telomeric 3= overhang. The template is then used to direct the iterative addition of the telomeric repeats (TTAGGG in humans), catalyzed by an active site within the TERT protein. Once TERT reaches the end of the RNA template, the DNA substrate is realigned so that additional repeats can be added (3). This ability of the RNA-protein enzyme complex to translocate underlies the enzyme's repeat-addition processivity (RAP). In addition to hTR and TERT, additional accessory proteins bind telomerase in vivo (4). Many studies have been done to identify important regions within hT...

show abstract

Section: Resultsmentioning

confidence: 97%

Section: (6)mentioning

confidence: 99%

Physical Connectivity Mapping by Circular Permutation of Human Telomerase RNA Reveals New Regions Critical for Activity and Processivity

Mefford

Zappulla

2016

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…The telomerase activation domain consists of the stem loops P6a/b, P6.1, and P5 (15) and is coordinated by the VSR motif of TRBD and motifs E-II and E-III (FVYL pocket) of the thumb domain (6,9,10). Although CR4/5 is primarily coordinated by the TRBD, its stem loop P6.1 extends across the TRBD-thumb interface and binds the FVYL pocket of the thumb domain (6).…”

mentioning

confidence: 99%

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

Hoffman

Rice

Skordalakes

2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

“…1B) (33). A solution NMR structure of the medaka TR (mdTR) activating domain (CR4/5) and the crystal structure of the CR4/5-TRBD complex have been reported (34,35). These structures show that phylogenetically conserved bases interact with the TRBD, suggesting that the mode of interaction is conserved among vertebrates (34).…”

Section: Significancementioning

confidence: 99%

“…A solution NMR structure of the medaka TR (mdTR) activating domain (CR4/5) and the crystal structure of the CR4/5-TRBD complex have been reported (34,35). These structures show that phylogenetically conserved bases interact with the TRBD, suggesting that the mode of interaction is conserved among vertebrates (34). Based on sequence comparisons, the medaka t/PK domain is predicted to have the minimal pseudoknot and P2ab, but not P2a.1, and its J2a/3 loop is much longer than in hTR (Fig.…”

Section: Significancementioning

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.

View full text Add to dashboard Cite

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 (...

show abstract

Structural basis for protein-RNA recognition in telomerase

Cited by 73 publications

References 38 publications

Physical Connectivity Mapping by Circular Permutation of Human Telomerase RNA Reveals New Regions Critical for Activity and Processivity

Physical Connectivity Mapping by Circular Permutation of Human Telomerase RNA Reveals New Regions Critical for Activity and Processivity

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

Contact Info

Product

Resources

About