Telomerase RNAs Research Articles

A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.

Cellular and Gene Expression Responses Involved in the Rapid Growth Inhibition of Human Cancer Cells by RNA Interference-mediated Depletion of Telomerase RNA

Inhibition of the up-regulated telomerase activity in cancer cells has previously been shown to slow cell growth but only after prior telomere shortening. Previously, we have reported that, unexpectedly, a hairpin short interfering RNA specifically targeting human telomerase RNA rapidly inhibits the growth of human cancer cells independently of p53 or telomere length and without bulk telomere shortening (Li, S., Rosenberg, J. E., Donjacour, A. A., Botchkina, I. L., Hom, Y. K., Cunha, G. R., and Blackburn, E. H. (2004) Cancer Res. 64, 4833-4840). Here we have demonstrated that such telomerase RNA knockdown in cancer cells does not cause telomere uncapping but rather induces changes in the global gene expression profile indicative of a novel response pathway, which includes suppression of specific genes implicated in angiogenesis and metastasis, and is distinct from the expression profile changes induced by telomere-uncapping mutant template telomerase RNAs. These cellular responses to depleting telomerase in human cancer cells together suggest that cancer cells are "telomerase-addicted" and uncover functions of telomerase in tumor growth and progression in addition to telomere maintenance.

The structure of an enzyme-activating fragment of human telomerase RNA

The ribonucleoprotein enzyme telomerase ensures the stability and fidelity of linear chromosome ends by elongating the telomeric DNA that is lost during each round of DNA replication. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence, acts as the scaffold for ribonucleoprotein assembly, and activates the enzyme for catalysis. Vertebrate telomerase RNAs contain three highly conserved structural and functional domains: the template domain, the "CR4-CR5" or "activation" domain essential for activation of the enzymatic activity, and a 3'-terminal "box H/ACA"-homology domain responsible for ribonucleprotein assembly and maturation. Here we report the NMR structure of a functionally essential RNA structural element derived from the human telomerase RNA CR4-CR5 domain. This RNA, referred to as hTR J6, forms a stable hairpin interrupted by a single nucleotide bulge and an asymmetric internal loop. Previous work on telomerase has shown that deletion of the hTR J6 asymmetric internal loop results in an RNA incapable of binding the enzymatic protein component of the RNP and therefore an inactive RNP without telomerase activity. We demonstrate here that the J6 internal loop introduces a twist in the RNA structure that may position the entire domain into the catalytic site of the enzyme.

The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle.

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1–871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1–RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1–RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1–RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.

An emerging consensus for telomerase RNA structure.

Telomerase maintains telomere length by adding G-rich telomeric repeats to the ends of linear eukaryotic chromosomes. The core telomerase enzyme consists of two components: an essential RNA component and a catalytic protein component. The catalytic component, telomerase reverse transcriptase (TERT), contains sequence motifs homologous to those in the catalytic domain of reverse transcriptase enzymes. Although the TERT component of telomerase is fairly conserved among eukaryotes, the RNA component varies dramatically in sequence composition and in size. The structure of the telomerase RNA has been well established in ciliates and vertebrates (1–4). These structural models have led to experiments that provided fundamental insight into telomerase RNA function. However, it has been difficult to determine the global secondary structure of the very large yeast RNA. In the past month, there have been four new reports proposing a secondary structure for the yeast telomerase RNA (5–8). In this issue of PNAS, Blackburn and colleagues (7) propose a core structure for the yeast telomerase RNA that resembles the structure present in ciliate and vertebrate telomerase RNAs. Telomerase RNA is very divergent across eukaryotic species. The size ranges from ≈150 nt in ciliates to >1,300 nt in fungi. Although there is some sequence similarity within each group of closely related eukaryotes, there is no sequence similarity between groups. Thus, the telomerase RNA sequences from vertebrates cannot be aligned with the RNA sequences from ciliates. The rapid divergence in RNA size and sequence has made it difficult to identify common secondary structures for telomerase RNAs. Phylogenetic comparative analysis has proven to be the most powerful approach for inferring higher-order RNA structures. In this method, base paired helices in RNA secondary structure are determined by nucleotide covariation between species (9). For example, an A:U base pair in one species may change to a …

A universal telomerase RNA core structure includes structured motifs required for binding the telomerase reverse transcriptase protein.

Telomerase synthesizes telomeric DNA by copying a short template sequence within its telomerase RNA component. We delineated nucleotides and base-pairings within a previously mapped central domain of the Saccharomyces cerevisiae telomerase RNA (TLC1) that are important for telomerase function and for binding to the telomerase catalytic protein Est2p. Phylogenetic comparison of telomerase RNA sequences from several budding yeasts revealed a core structure common to Saccharomyces and Kluyveromyces yeast species. We show that in this structure three conserved sequences interact to provide a binding site for Est2p positioned near the template. These results, combined with previous studies on telomerase RNAs from other budding yeasts, vertebrates, and ciliates, define a minimal universal core for telomerase RNAs.

A Phylogenetically Based Secondary Structure for the Yeast Telomerase RNA

Background: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure.Results: We cloned and analyzed the complete telomerase RNA loci (TLC1) from all known Saccharomyces species belonging to the “sensu stricto” group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core.Conclusions: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.

Functional organization of repeat addition processivity and DNA synthesis determinants in the human telomerase multimer.

Human telomerase is a multimer containing two human telomerase RNAs (hTRs) and most likely two human telomerase reverse transcriptases (hTERTs). Telomerase synthesizes multiple telomeric repeats using a unique repeat addition form of processivity. We investigated hTR and hTERT sequences that were essential for DNA synthesis and processivity using a direct primer extension telomerase assay. We found that hTERT consists of two physically separable functional domains, a polymerase domain containing RNA interaction domain 2 (RID2), reverse transcriptase (RT), and C-terminal sequences, and a major accessory domain, RNA interaction domain 1 (RID1). RID2 mutants defective in high-affinity hTR interactions and an RT catalytic mutant exhibited comparable DNA synthesis defects. The RID2-interacting hTR P6.1 helix was also essential for DNA synthesis. RID1 interacted with the hTR pseudoknot-template domain and hTERT's RT motifs and putative thumb and was essential for processivity, but not DNA synthesis. The hTR pseudoknot was essential for processivity, but not DNA synthesis, and processivity was reduced or abolished in dimerization-defective pseudoknot mutants. trans-acting hTERTs and hTRs complemented the processivity defects of RID1 and pseudoknot mutants, respectively. These data provide novel insight into the catalytic organization of the human telomerase complex and suggest that repeat addition processivity is one of the major catalytic properties conferred by telomerase multimerization.

Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins

Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10. The assembly and molecular interactions of the components of H/ACA RNPs are unknown. Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following. NOP10 associates with NAP57 as a prerequisite for NHP2 binding. Although NHP2 on its own binds RNA nonspecifically, this NAP57-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs. GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs. In contrast to other RNPs whose assembly is initiated by protein-RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs. Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis.

A novel pseudoknot element is essential for the action of a yeast telomerase.

Telomerase contains an essential RNA, which includes the template sequence copied by the reverse transcription action of telomerase into telomeric DNA. Using phylogenetic comparison, we identified seven conserved sequences in telomerase RNAs from Kluyveromyces budding yeasts. We show that two of these sequences, CS3 and CS4, are essential for normal telomerase function and can base-pair to form a putative long-range pseudoknot. Disrupting this base-pairing was deleterious to cell growth, telomere maintenance, and telomerase activity. Restoration of the base-pairing potential alleviated these phenotypes. Mutating this pseudoknot caused a novel mode of shifting of the boundaries of the RNA template sequence copied by telomerase. A phylogenetically derived model of yeast TER structure indicates that these RNAs can form two alternative predicted core conformations of similar stability: one brings the CS3/CS4 pseudoknot spatially close to the template; in the other, CS3 and CS4 move apart and the conformation of the template is altered. We propose that such disruption of the pseudoknot, and potentially the predicted telomerase RNA conformation, affects polymerization to cause the observed shifts in template usage.

The solution structure of an essential stem-loop of human telomerase RNA.

The ribonucleoprotein enzyme telomerase maintains chromosome ends in most eukaryotes and is critical for a cell's genetic stability and its proliferative viability. All telomerases contain a catalytic protein component homologous to viral reverse transcriptases (TERT) and an RNA (TR) that provides the template sequence as well as a scaffold for ribonucleoprotein assembly. Vertebrate telomerase RNAs have three essential domains: the template, activation and stability domains. Here we report the NMR structure of an essential RNA element derived from the human telomerase RNA activation domain. The sequence forms a stem-loop structure stabilized by a GU wobble pair formed by two of the five unpaired residues capping a short double helical region. The remaining three loop residues are in a well-defined conformation and form phosphate-base stacking interactions reminiscent of other RNA loop structures. Mutations of these unpaired nucleotides abolish enzymatic activity. The structure rationalizes a number of biochemical observations, and allows us to propose how the loop may function in the telomerase catalytic cycle. The pre-formed structure of the loop exposes the bases of these three essential nucleotides and positions them to interact with other RNA sequences within TR, with the reverse transcriptase or with the newly synthesized telomeric DNA strand. The functional role of this stem-loop appears to be conserved in even distantly related organisms such as yeast and ciliates.

A bulged stem tethers Est1p to telomerase RNA in budding yeast.

It is well established that the template for telomeric DNA synthesis is provided by the RNA subunit of telomerase; however, the additional functions provided by most of the rest of the RNA (>1000 nucleotides in budding yeast) are largely unknown. By alignment of telomerase RNAs of Saccharomyces cerevisiae and six Kluyveromyces species followed by mutagenesis of the S. cerevisiae RNA, we found a conserved region that is essential for telomere maintenance. Phylogenetic analysis and computer folding revealed that this region is conserved not only in primary nucleotide sequence but also in secondary structure. A common bulged-stem structure was predicted in all seven yeast species. Mutational analysis showed the structure to be essential for telomerase function. Suppression of bulged-stem mutant phenotypes by overexpression of Est1p and loss of co-immunoprecipitation of the mutant RNAs with Est1p indicated that this bulged stem is necessary for association of Est1p, a telomerase regulatory subunit. Est1p in yeast extract bound specifically to a small RNA containing the bulged stem, suggesting a direct interaction. We propose that this RNA structure links the enzymatic core of telomerase with Est1p, thereby allowing Est1p to recruit or activate telomerase at the telomere.

Phylogenetic relationships amongst tetrahymenine ciliates inferred by a comparison of telomerase RNAs

Phylogenetic relationships amongst tetrahymenine ciliates inferred by a comparison of telomerase RNAs

A critical stem-loop structure in the CR4-CR5 domain of mammalian telomerase RNA.

Telomerase is an enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The telomerase ribonucleoprotein complex consists of two essential components, a reverse transcriptase and an RNA molecule that provides the template for telomeric repeat synthesis. A common secondary structure of vertebrate telomerase RNA has been proposed based on a phylogenetic comparative analysis of 35 sequences. Here we report the identification of an additional essential base-paired region in the CR4-CR5 domain of mammalian telomerase RNA, termed P6.1. Mouse telomerase RNAs with mutations that disrupted base pairings in the P6.1 helix were unable to reconstitute telomerase activity in vivo. In contrast, an RNA mutant with compensatory mutations that restored base pairings in the P6.1 helix restored telomerase activity. In an in vitro reconstitution system stable base pairing of the P6.1 stem was required for the RNA-protein interaction between the CR4-CR5 domain and the telomerase reverse transcriptase (TERT) protein. Interestingly, two RNA mutations, one that extends the P6.1 stem and one that alters the conserved nucleotides of the L6.1 loop, allowed RNA-protein binding but significantly impaired telomerase activity. These data establish the presence of the P6.1 stem-loop and its importance for the assembly and enzymatic activity of the mammalian telomerase complex.

Human telomerase RNA-protein interactions.

Telomere length is maintained in most eukaryotic cells by telomerase. The core components of this ribonucleoprotein (RNP) enzyme include a protein catalytic subunit, composed of motifs conserved among reverse transcriptases (RT), and an RNA subunit that contains a short template sequence essential for the synthesis of telomeric repeats. We developed an electrophoretic mobility shift assay using active telomerase partially purified from 293 cells and radiolabeled, in vitro-transcribed human telomerase RNA (hTR) to investigate the molecular interactions of the human telomerase RT (hTERT) and telomerase-associated proteins with hTR. A specific hTR-protein complex was identified and shown to contain hTERT and human Staufen by antibody supershift assays. Variants of hTR altered in distinct structural elements were analyzed for their ability to competitively inhibit complex formation. Human telomerase RNAs lacking the CR4-CR5 domain were poor inhibitors of hTR-protein complex formation, suggesting that the CR4-CR5 domain of hTR is a potential protein-binding site. Furthermore, alterations in the telomerase RNA pseudoknot's P3 helix, the CR7 domain, or the H/ACA box efficiently inhibited formation of the complex, indicating that these domains are dispensable for the assembly of a telomerase RNP in vitro. Potential telomerase-associated proteins that bind hTR were also identified using a UV cross-linking assay.

A low threshold level of expression of mutant-template telomerase RNA inhibits human tumor cell proliferation.

The ribonucleoprotein telomerase synthesizes telomeric DNA by copying an intrinsic RNA template. In most cancer cells, telomerase is highly activated. Here we report a telomerase-based antitumor strategy: expression of mutant-template telomerase RNAs in human cancer cells. We expressed mutant-template human telomerase RNAs in prostate (LNCaP) and breast (MCF-7) cancer cell lines. Even a low threshold level of expression of telomerase RNA gene constructs containing various mutant templates, but not the control wild-type template, decreased cellular viability and increased apoptosis. This occurred despite the retention of normal levels of the endogenous wild-type telomerase RNA and endogenous wild-type telomerase activity and unaltered stable telomere lengths. In vivo tumor xenografts of a breast cancer cell line expressing a mutant-template telomerase RNA also had decreased growth rates. Therefore, mutant-template telomerase RNAs exert a strongly dominant-negative effect on cell proliferation and tumor growth. These results support the potential use of mutant-template telomerase RNA expression as an antineoplastic strategy.

Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability.

We have reconstituted wild type or mutant telomerase activity in two human cell lines that lack constitutive expression of both core subunits of the enzyme and maintain telomeres by a telomerase-independent mechanism (ALT cells). Wild type telomerase RNA and four telomerase RNAs with single point mutations in their template domain were used to express enzymes specifying different telomeric DNA sequences. Expression of wild type telomerase for up to 32 days had no detectable effect on cell growth or viability. In contrast, cells expressing mutant telomerases had slower growth rate, abnormal cell cycle and reduced viability. Dramatically aberrant nuclei, typical of cells undergoing mitotic catastrophe, and large numbers of fused chromosomes were also characteristic of these populations. Notably, all phenotypes were apparent within the first few cell divisions after expression of the enzymes. Unlike wild type, mutant telomerase activity was progressively selected against with cell culturing, and this correlated with the disappearance of cells with aberrant phenotypes. Our results suggest that even very limited synthesis of mutated sequences can affect telomere structure in human cells, and that the toxicity of mutant telomerases is due to telomere malfunction.

In vitro assembly of human H/ACA small nucleolar RNPs reveals unique features of U17 and telomerase RNAs.

The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3'-terminal stem-loop followed by box ACA (U17/3'st) was sufficient to form an RNP, and U17/3'st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3' moiety of human telomerase RNA (hTR), and its 3'-terminal stem-loop (hTR/3'st), also could form an RNP by binding H/ACA proteins. Hence, the 3'-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins.

Expression of mutated Paramecium telomerase RNAs in vivo leads to templating errors that resemble those made by retroviral reverse transcriptase.

Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutated P. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.

A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end.

Simple sequence repeat telomeric DNA is maintained by a specialized reverse transcriptase, telomerase. The integral RNA subunit of telomerase contains a template region that determines the sequence added to chromosome ends. Aside from providing the template, little is known about the role of the telomerase RNA. In addition, no hypotheses have been suggested to account for the striking evolutionary divergence in size and sequence between telomerase RNAs of ciliates, yeasts, and mammals. We show that the two- to threefold increase in size of the mammalian telomerase RNAs relative to ciliate telomerase RNAs is due to the presence of an extra domain resembling a box H/ACA small nucleolar RNA (snoRNA). The human telomerase RNA (hTR) H/ACA domain is essential in vivo for hTR accumulation, hTR 3' end processing, and telomerase activity. By substituting the U64 box H/ACA snoRNA for the hTR H/ACA domain, we demonstrate that a heterologous snoRNA can function to promote chimeric RNA accumulation and 3' end processing but not telomerase activity. In addition, we show that maturation of full-length hTR and its assembly into active telomerase occur from an mRNA promoter-driven RNA polymerase II transcript but not from a U6 snRNA promoter-driven RNA polymerase III transcript. Finally, we show that a small percentage of hTR is associated with nucleoli. These results have implications for the biogenesis and structure of hTR and the human telomerase ribonucleoprotein complex. They also expand the structural and functional diversity of the box H/ACA snoRNA motif.