Solution Structure and Dynamics of the Wild-type Pseudoknot of Human Telomerase RNA

Kim, Nak Kyoon; Zhang, Qi; Zhou, Jing; Theimer, Carla A.; Peterson, Robert D.; Feigon, Juli

doi:10.1016/j.jmb.2008.10.005

Cited by 84 publications

(169 citation statements)

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“…Previous NMR studies suggested that an isolated pseudoknot sequence from the human telomerase RNA slowly interconverts between a hairpin state and the pseudoknot conformation on the timescale that is longer than seconds (22,33), whereas mutational analysis of human telomerase supports a static pseudoknot structure (25). In the single-molecule time traces shown here, neither stem A nor stem B were observed to undergo conformational rearrangements in the wild-type pseudoknot during primer extension or enzyme translocation.…”

Section: Discussionmentioning

confidence: 55%

“…Phylogenetic analyses of ciliate (9,17), yeast (18)(19)(20), and vertebrate (21) RNAs suggest that this region may fold into a pseudoknot structure. NMR and single-molecule force studies of the isolated pseudoknot sequence from human show that this sequence can indeed form a pseudoknot structure (22,23). Mutation analyses indicate that disruption of potential base pairs in the motif correlates with telomerase assembly and activity defects in multiple organisms (18,24,25), but in vitro telomerase activity assays give varying results on the functional importance of the motif (12,13,(26)(27)(28).…”

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confidence: 99%

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Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Mihalusova¹,

Wu²,

Zhuang

2011

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

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Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.T elomeres shorten with each round of DNA replication. Once telomeres reach a critical length, they become vulnerable to DNA damage response, which triggers cellular senescence and chromosome fusion (1). Telomerase protects telomeres from this replication-induced DNA erosion by addition of short G-rich repeats to the DNA ends (2). The telomerase enzyme is an attractive drug target in both cancer and regenerative medicine, because most highly proliferative cells, including stem and cancer cells, rely on its activity to maintain genomic integrity (3). Furthermore, multiple human diseases are known to be caused by mutations in telomerase subunits (4-6), underscoring the importance of understanding the enzyme's structure and function.The ciliate Tetrahymena thermophila has long been used as a model system to study telomerase activity. Tetrahymena telomerase is comprised of a 159-nucleotide RNA subunit, a 133-kDa telomerase reverse transcriptase (TERT), and multiple protein cofactors (7). The RNA has a conserved secondary structure (Fig. 1A) that contains a template region, which serves as template for the telomeric repeat synthesis, flanked upstream by the template boundary element (TBE) and downstream by a putative pseudoknot, as well as a highly structured region made of stems I and IV (8-10). TERT, the catalytic subunit, binds telomerase RNA in the TBE region, the template recognition element, and the loop of stem IV (10-13) (Fig. 1A) . Of the multiple Tetrahymena telomerase cofactors identified to date, only p65 binds the RNA directly in the stem I-proximal stem IV region (Fig. 1A) and promotes the hierarchical telomerase RNP assembly (14-16).Past studies have suggested that the putative pseudoknot region downstream of the template is a conserved and functionally important segment of telomerase RNA. Phylogenetic analyses of ciliate (9, 17), yeast (18-20), and vertebrate (21) RNAs suggest that this region may fold into a pse...

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Section: Discussionmentioning

confidence: 55%

mentioning

confidence: 99%

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Mihalusova¹,

Wu²,

Zhuang

2011

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

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“…The assignments of preQ 1 and interactions in the binding pocket were identified as described previously (33). Single-bond C-H and N-H RDCs were measured using 2D 1 H-13 C S3CT-HSQC and standard 1 H-15 N HSQC experiments as described previously (70), and are listed in Table S3.…”

Section: Methodsmentioning

confidence: 99%

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Kang

Eichhorn²,

Feigon

2014

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

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Prequeuosine (preQ 1 ) riboswitches are RNA regulatory elements located in the 5′ UTR of genes involved in the biosynthesis and transport of preQ 1 , a precursor of the modified base queuosine universally found in four tRNAs. The preQ 1 class II (preQ 1 -II) riboswitch regulates preQ 1 biosynthesis at the translational level. We present the solution NMR structure and conformational dynamics of the 59 nucleotide Streptococcus pneumoniae preQ 1 -II riboswitch bound to preQ 1 . Unlike in the preQ 1 class I (preQ 1 -I) riboswitch, divalent cations are required for high-affinity binding. The solution structure is an unusual H-type pseudoknot featuring a P4 hairpin embedded in loop 3, which forms a three-way junction with the other two stems. 13 C relaxation and residual dipolar coupling experiments revealed interhelical flexibility of P4. We found that the P4 helix and flanking adenine residues play crucial and unexpected roles in controlling pseudoknot formation and, in turn, sequestering the Shine-Dalgarno sequence. Aided by divalent cations, P4 is poised to act as a "screw cap" on preQ 1 recognition to block ligand exit and stabilize the binding pocket.Comparison of preQ 1 -I and preQ 1 -II riboswitch structures reveals that whereas both form H-type pseudoknots and recognize preQ 1 using one A, C, or U nucleotide from each of three loops, these nucleotides interact with preQ 1 differently, with preQ 1 inserting into different grooves. Our studies show that the preQ 1 -II riboswitch uses an unusual mechanism to harness exquisite control over queuosine metabolism.R iboswitches are functional noncoding RNA elements often found at the 5′ end of genes that bind to effector molecules, usually metabolites, to attenuate gene expression (1-8). They generally contain two modular elements, an aptamer that binds directly to a cognate ligand, followed by an expression platform that carries out the regulatory function. Ligand binding induces a conformational rearrangement in the aptamer domain, which is transduced to the expression platform, turning gene expression on or off at the transcription or translation level.Structural studies of diverse riboswitches bound to their cognate ligands have revealed that they have evolved various strategies for high-affinity and specific binding between RNA receptors and ligands. In general, the aptamer domain enfolds the ligand, forming a highly complex 3D conformation using a host of tertiary interactions, including base triples, kink turns, ribose zippers, A-minor motifs, loop-loop interactions, and pseudoknots (9-11). Some riboswitches require divalent cations for ligand binding (9, 12-16), whereas in others divalent cations stabilize the bound state (17-21). Some ligands, including S-adenosyl methionine (22), S-adenosyl homocysteine (22, 23), cyclic di-GMP (24, 25), and prequeuosine (preQ 1 ) (26, 27), have more than one class of riboswitch, with each class adopting a different 3D fold to achieve recognition of the same ligand.Two classes of riboswitches that bind preQ 1 , a precursor t...

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“…TER pseudoknot domain (nt contains the telomeric repeat template [91]. Together with the TER CR4-CR5 domain (nt , the pseudoknot domain is also necessary for the binding of TERT and optimal enzyme processivity [54, 92,93]. The hairpin-hinge-hairpin-ACA (H/ACA) motif of TER at nt 275-451 is critical for its cellular accumulation, 3′ end processing, and intranuclear trafficking [58,94].…”

Section: Telomerase Rna (Ter)mentioning

confidence: 99%

Genetic Variations in Telomere Maintenance, with Implications on Tissue Renewal Capacity and Chronic Disease Pathologies

Trudeau¹,

Wong²

2010

CPPM

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Premature loss of telomere repeats underlies the pathologies of inherited bone marrow failure syndromes. Over the past decade, researchers have mapped genetic lesions responsible for the accelerated loss of telomere repeats. Haploinsufficiencies in the catalytic core components of the telomere maintenance enzyme telomerase, as well as genetic defects in telomerase holoenzyme components responsible for enzyme stability, have been linked to hematopoietic failure pathologies. Frequencies of these disease-associated alleles in human populations are low. Accordingly, the diseases themselves are rare. On the other hand, single nucleotide polymorphisms of telomerase enzyme components are found with much higher frequencies, with several non-synonymous SNP alleles observed in 2-4% of the general population. Importantly, recent advents of molecular diagnostic techniques have uncovered links between telomere length maintenance deficiencies and an increasing number of pathologies unrelated to the hematopoietic system. In these cases, short telomere length correlates to tissue renewal capacities and predicts clinical progression and disease severity. To the authors of this review, these new discoveries imply that even minor genetic defects in telomere maintenance can culminate in the premature failure of tissue compartments with high renewal rates. In this review, we discuss the biology and molecules of telomere maintenance, and the pathologies associated with an accelerated loss of telomeres, along with their etiologies. We also discuss single nucleotide polymorphisms of key telomerase components and their association with tissue renewal deficiency syndromes and other pathologies. We suggest that inter-individual variability in telomere maintenance capacity could play a significant role in chronic inflammatory diseases, and that this is not yet fully appreciated in the translational research of pharmacogenomics and personalized medicine.

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Solution Structure and Dynamics of the Wild-type Pseudoknot of Human Telomerase RNA

Cited by 84 publications

References 59 publications

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Structural determinants for ligand capture by a class II preQ ₁ riboswitch

Genetic Variations in Telomere Maintenance, with Implications on Tissue Renewal Capacity and Chronic Disease Pathologies

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Solution Structure and Dynamics of the Wild-type Pseudoknot of Human Telomerase RNA

Cited by 84 publications

References 59 publications

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Structural determinants for ligand capture by a class II preQ 1 riboswitch

Genetic Variations in Telomere Maintenance, with Implications on Tissue Renewal Capacity and Chronic Disease Pathologies

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Structural determinants for ligand capture by a class II preQ ₁ riboswitch