Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA

Kim, Nak-Kyoon; Theimer, Carla A.; Mitchell, James R.; Collins, Kathleen; Feigon, Juli

doi:10.1093/nar/gkq525

Cited by 65 publications

(75 citation statements)

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“…Truncations of CR4/5 RNA showed stem P5 is not necessary for TRBD binding (Fig. S7B, mutants M10-11), in accordance with a previous study that also showed P5 is dispensable for reconstituting telomerase activity in vitro (26). The minimal RNA fragment that retained wild-type binding affinity is only 39 nt, spanning residue 176 to residue 214.…”

Section: Resultssupporting

confidence: 90%

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: Resultssupporting

confidence: 90%

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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“…In addition, a minimal CR4/CR5 domain construct, which includes P6.1, all of P6a-P6b except the terminal hairpin, and flanking nucleotides but does not include the P5 helix, is sufficient to reconstitute telomerase activity in trans with the core domain and TERT (Fig. 5A) (76). Thus, it is not clear whether this region forms a three-way junction in the context of its association with TERT.…”

Section: Ste Of Htrmentioning

confidence: 99%

“…To date, there are no crystal structures of telomerase RNA or telomerase protein-RNA complexes; however, over the last several years, structures of several key hTR elements have been determined by NMR spectroscopy (52,54,55,60,(74)(75)(76). This review summarizes what NMR structural and dynamics studies, combined with biochemical and mutational analysis, have revealed about the functional roles of hTR domains and the overall architecture of hTR.…”

mentioning

confidence: 99%

Architecture of human telomerase RNA

Zhang

Kim

Feigon

2011

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

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147

129

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Telomerase is a unique reverse transcriptase that catalyzes the addition of telomere DNA repeats onto the 3′ ends of linear chromosomes and plays a critical role in maintaining genome stability. Unlike other reverse transcriptases, telomerase is unique in that it is a ribonucleoprotein complex, where the RNA component [telomerase RNA (TR)] not only provides the template for the synthesis of telomere DNA repeats but also plays essential roles in catalysis, accumulation, TR 3′-end processing, localization, and holoenzyme assembly. Biochemical studies have identified TR elements essential for catalysis that share remarkably conserved secondary structures across different species as well as species-specific domains for other functions, paving the way for high-resolution structure determination of TRs. Over the past decade, structures of key elements from the core, conserved regions 4 and 5, and small Cajal body specific RNA domains of human TR have emerged, providing significant insights into the roles of these RNA elements in telomerase function. Structures of all helical elements of the core domain have been recently reported, providing the basis for a high-resolution model of the complete core domain. We review this progress to determine the overall architecture of human telomerase RNA.Box H/ACA RNA | NMR | pseudoknot | telomerase reverse transcriptase T elomerase is a large, multisubunit ribonucleoprotein (RNP) that replicates the 3′ end of linear chromosomes by processive synthesis of telomere DNA repeats. Telomeres, the physical ends of linear chromosomes, generally comprise dsDNA with a short repeating species-specific sequence ending in a 3′ single-stranded overhang of variable length plus associated telomere binding proteins, called shelterin in humans (1, 2). Telomeres protect the integrity of linear chromosomes by allowing the cellular DNA repair machinery to distinguish them from double-strand breaks, thus playing critical roles in maintaining genome stability in eukaryotes (1, 2). Shortening of telomeres below a critical length because of inherent incomplete replication of DNA ends ultimately leads to telomere fusions and cell senescence (3-6). The 3′ ends of telomeres are replicated by telomerase, a unique reverse transcriptase discovered almost three decades ago (7), which catalyzes the addition of telomere DNA repeats onto the ends of linear chromosomes using an embedded RNA as the template (8, 9). Although telomerase has a low or undetectable level of activity in most somatic cells, it is active in some germline, epithelial, and hematopoietic cells, and it is highly active in the majority (∼90%) of cancer cell lines (10-12). Telomerase deficiency because of mutations in human telomerase RNA (hTR) has also been linked to several inherited human diseases, such as dyskeratosis congenita, aplastic anemia, myelodysplasia, and idiopathic pulmonary fibrosis (13-26).The telomerase holoenzyme includes a unique reverse transcriptase [telomerase reverse transcriptase (TERT)], an essential RNA (TR), and several...

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“…Only the structures of isolated elements have been solved by NMR (Leeper et al 2003;Leeper and Varani 2005;Theimer et al 2005Theimer et al , 2007Chen et al 2006;Richards et al 2006a,b;Kim et al 2008Kim et al , 2010Kim et al , 2014Zhang et al 2010Zhang et al , 2011. Although there have been some previous chemical structure-probing analyses of telomerase RNAs (Antal et al 2002;Forstemann and Lingner 2005), even secondary-structure models have been difficult to validate or refine.…”

Section: Introductionmentioning

confidence: 99%

Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

Niederer

Zappulla

2014

RNA

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Telomerase catalyzes the addition of nucleotides to the ends of chromosomes to complete genomic DNA replication in eukaryotes and is implicated in multiple diseases, including most cancers. The core enzyme is composed of a reverse transcriptase and an RNA subunit, which provides the template for DNA synthesis. Despite extensive divergence at the sequence level, telomerase RNAs share several structural features within the catalytic core, suggesting a conserved enzyme mechanism. We have investigated the structure of the core of the human and yeast telomerase RNAs using SHAPE, which interrogates flexibility of each nucleotide. We present improved secondary-structure models, refined by addition of five base triples within the yeast pseudoknot and an alternate pairing within the human-specific element J2a.1 in the human pseudoknot, both of which have implications for thermodynamic stability. We also identified a potentially structured CCC region within the template that may facilitate substrate binding and enzyme mechanism. Overall, the SHAPE findings reveal multiple similarities between the Saccharomyces cerevisiae and Homo sapiens telomerase RNA cores.

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Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA

Cited by 65 publications

References 61 publications

RNA–protein binding interface in the telomerase ribonucleoprotein

RNA–protein binding interface in the telomerase ribonucleoprotein

Architecture of human telomerase RNA

Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE

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