Functional Multimerization of Human Telomerase Requires an RNA Interaction Domain in the N Terminus of the Catalytic Subunit

Moriarty, Tara J.; Huard, Sylvain; Dupuis, Sophie; Autexier, Chantal

doi:10.1128/mcb.22.4.1253-1265.2002

Cited by 106 publications

(147 citation statements)

References 45 publications

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“…S8C). Deletion of this motif completely abolishes telomerase activity, whereas RNA binding is only slightly reduced (9). The vertebrate-specific motif VSR contains the third cross-linked residue Phe355 and is part of the CR4/5 binding pocket.…”

Section: Discussionmentioning

confidence: 99%

“…The vertebrate TRBD contains an additional vertebrate-specific RNA binding (VSR) motif (9). Substitution and deletion studies found CP, QFP, T, and VSR motifs are required for CR4/5 binding in vertebrates (9,13,14). In budding yeast, the TR contains a conserved three-way junction structure that is similar to the vertebrate CR4/5 domain but does not appear to bind TRBD in vitro (15).…”

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confidence: 99%

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confidence: 99%

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RNA–protein binding interface in the telomerase ribonucleoprotein

Bley¹,

Qi²,

Rand³

et al. 2011

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

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Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR-TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA-protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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RNA–protein binding interface in the telomerase ribonucleoprotein

Bley¹,

Qi²,

Rand³

et al. 2011

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

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“…Being the catalyst interacting with both hTERC and telomeric DNA, hTERT contains four major regions: an N-terminal regulatory (R) domain, RNA-binding (RB) domain, reverse transcription (RT) domain and C-terminal dimerization domain [45]. Several motifs in the R and RB domains of hTERT are conserved in different species, important for various molecular interactions [41,[46][47][48]. As the rate-limiting component of telomerase, hTERT induces immortalization of a number of cell types in culture [49] (reviewed in [50]).…”

Section: Telomeres Telomerase and Cell Immortalizationmentioning

confidence: 99%

Mechanisms of cell immortalization mediated by EB viral activation of telomerase in nasopharyngeal carcinoma

et al. 2006

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Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and Southeast Asia. The disease is a poorly differentiated carcinoma without effective cure, and the mechanism underlying its development remains largely unknown. Of several factors identified in NPC aetiology in recent years, Epstein-Barr virus (EBV) infection has emerged to be most important. In almost all NPC cells, EBV uses several intracellular mechanisms to cause oncogenic evolution of the infected cells. One such mechanism by which EBV infection induces cellular immortalization is believed to be through the activation of telomerase, an enzyme that is normally repressed but becomes activated during cancer development. Studies show that greater than 85% of primary NPC display high telomerase activity by mechanisms involving EBV infection, consistent with the notion that EBV is commonly involved in inducing cell immortalization. More recently, different EBV proteins have been shown to activate or inhibit the human telomerase reverse transcriptase gene, by modulating intracellular signalling pathways. These findings suggest a new model with a number of challenges towards our understanding, molecular targeting and therapeutic intervention in NPC.

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“…Human telomerase enzyme functions as a dimer (17)(18)(19). However, the RNA conformation within the telomerase dimer complex is not well studied.…”

Section: In Vivo Confirmation Of the Pseudoknot Structure And Functionmentioning

confidence: 99%

Functional analysis of the pseudoknot structure in human telomerase RNA

Chen

Greider

2005

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

122

107

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Telomerase is essential for maintaining telomere length and chromosome stability in stem cells, germline cells, and cancer cells. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein component and an RNA molecule that provides the template for telomeric repeat synthesis. A pseudoknot structure in the human telomerase RNA is conserved in all vertebrates and is essential for telomerase activity. It has been proposed that this highly conserved structure functions as a dynamic structure with conformational interchange between the pseudoknot and a hairpin with intraloop base pairings. To examine the structural and functional requirements of the pseudoknot structure, we made mutations in the proposed base-paired regions in the pseudoknot. Although mutations that disrupted the pseudoknot P3 helix abolished activity as predicted, mutations that disrupted the intraloop hairpin base pairings did not reduce telomerase activity, indicating that the intraloop hairpin is not required for telomerase function. This functional study thus provides evidence against the previous proposed molecular-switch model of telomerase pseudoknot function and supports a static pseudoknot structure. The mutational analysis further suggests that telomerase RNA can function independent of the proposed intermolecular pairings between pseudoknot regions on two RNA molecules.reverse transcriptase ͉ ribonucleoprotein ͉ telomere T elomerase is a unique ribonucleoprotein complex that is essential for telomere maintenance. Vertebrate telomeres are required for the stable chromosome maintenance; they consist of simple tandemly repeated TTAGGG sequences and telomere-associated proteins. Telomerase maintains telomeres by adding telomeric repeats to chromosome ends to counterbalance the natural shortening that occurs during DNA replication. Telomerase consists of two essential core components, the catalytic protein component telomerase reverse transcriptase (TERT), and the telomerase RNA (TR) that specifies the repeat sequence added. Vertebrate TR comprises three highly conserved structural domains: the template͞pseudoknot domain, CR4-CR5 domain, and the small Cajal-body RNA domain (1-4). The template͞pseudoknot domain contains the template region for telomeric DNA synthesis and a conserved pseudoknot structure essential for telomerase activity (Fig. 1A). The template region and the pseudoknot structure are brought into proximity by long-range base pairing interactions, i.e., the P1 helix. The integrity of the P1 helix plays an important role in template boundary definition (5). The presence of the pseudoknot structure formed by helices P2b and P3 was predicted based on phylogenetic analysis (1) and confirmed by mutational analysis (6). Although the pseudoknot structures are conserved in ciliate and vertebrate TRs, the molecular mechanism of their function in telomerase activity is unclear. A ''molecular switch'' hypothesis for pseudoknot function was proposed based on a NMR structure of the P2b stem-loop (Fig. ...

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Functional Multimerization of Human Telomerase Requires an RNA Interaction Domain in the N Terminus of the Catalytic Subunit

Cited by 106 publications

References 45 publications

RNA–protein binding interface in the telomerase ribonucleoprotein

RNA–protein binding interface in the telomerase ribonucleoprotein

Mechanisms of cell immortalization mediated by EB viral activation of telomerase in nasopharyngeal carcinoma

Functional analysis of the pseudoknot structure in human telomerase RNA

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