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Telomerase is a eukaryotic ribonucleoprotein (RNP) whose specialized reverse transcriptase action performs de novo synthesis of one strand of telomeric DNA. The resulting telomerase-mediated elongation of telomeres, which are the protective end-caps for eukaryotic chromosomes, counterbalances the inevitable attrition from incomplete DNA replication and nuclease action. The telomerase strategy to maintain telomeres is deeply conserved among eukaryotes, yet the RNA component of telomerase, which carries the built-in template for telomeric DNA repeat synthesis, has evolutionarily diverse size and sequence. Telomerase shows a distribution of labor between RNA and protein in aspects of the polymerization reaction. This article first describes the underlying conservation of a core set of structural features of telomerase RNAs important for the fundamental polymerase activity of telomerase. These include a pseudoknot-plus-template domain and at least one other RNA structural motif separate from the template-containing domain. The principles driving the diversity of telomerase RNAs are then explored. Much of the diversification of telomerase RNAs has come from apparent gain-of-function elaborations, through inferred evolutionary acquisitions of various RNA motifs used for telomerase RNP biogenesis, cellular trafficking of enzyme components, and regulation of telomerase action at telomeres. Telomerase offers broadly applicable insights into the interplay of protein and RNA functions in the context of an RNP enzyme.

The kinases ATM and ATR (Tel1 and Mec1 in the yeast Saccharomyces cerevisiae) control the response to DNA damage. We report that S. cerevisiae Tel2 acts at an early step of the TEL1/ATM pathway of DNA damage signaling. We show that Tel1 and Tel2 interact, and that even when Tel1 protein levels are high, this interaction is specifically required for Tel1 localization to a DNA break and its activation of downstream targets. Computational analysis revealed structural homology between Tel2 and Ddc2 (ATRIP in vertebrates), a partner of Mec1, suggesting a common structural principle used by partners of phoshoinositide 3-kinase-like kinases.

Telomeres are maintained by the ribonucleoprotein (RNP) enzyme telomerase, which replenishes telomeres through its unique mechanism of internal RNA-templated addition of telomeric DNA. Telomerase is active in most human cancers, typically because its core protein subunit, TERT, is up-regulated. Although the major known function of telomerase in cancer is to replenish telomeric DNA and maintain cell immortality, the regulation of the RNA component of telomerase is not well understood. In the course of investigations that have implicated telomerase RNA in key aspects of cancer progression, including metastasis, we explored some of the cis-acting elements affecting telomerase RNA expression and knockdown. The expression efficiency and subsequent RNA processing to produce the mature hTER differed considerably among various promoters. Together with other results, these findings establish that the crucial elements of the hTER gene affecting RNA-processing efficiency to produce the mature hTER RNA are the promoter and internal telomerase RNA-coding sequences.

Human TRF1-binding protein PinX1 inhibits telomerase activity. Here we report that overexpression of yeast PinX1p (yPinX1p) results in shortened telomeres and decreased in vitro telomerase activity. yPinX1p coimmunoprecipitated with yeast telomerase protein Est2p even in cells lacking the telomerase RNA TLC1, or the telomerase-associated proteins Est1p and Est3p. Est2p regions required for binding to yPinX1p or TLC1 were similar. Furthermore, we found two distinct Est2p complexes exist, containing either yPinX1p or TLC1. Levels of Est2p-yPinX1p complex increased when TLC1 was deleted and decreased when TLC1 was overexpressed. Hence, we propose that yPinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by 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 integral RNA (hTER) of the human telomerase ribonucleoprotein has a conserved secondary structure that contains a potential pseudoknot. Here we examine the role of an intermolecular hTER-hTER interaction in the previously reported functional dimerization of telomerase. We provide evidence that the two conserved, complementary sequences of one stem of the hTER pseudoknot domain can pair intermolecularly in vitro, and that formation of this stem as part of a novel "trans-pseudoknot" is required for telomerase to be active in its dimeric form. Such RNA-RNA interaction mirrors a known property of retroviral reverse transcriptases, which use homodimeric viral genomic RNA substrates.

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.

Multiple interacting components of the telomere, together with telomerase (and sometimes recombination), determine whether a telomere will be functional, allowing cell proliferation. The various components reinforce each other, providing for a robust and resilient system of protection and replenishment of telomeres. A characteristic of a telomere is that its structural features elicit responses that allow it to be dynamically recapped. Eliciting a DNA damage response through uncapping of a telomere appears to be one way in which telomerase action at that telomere is stimulated. Thus, as long as a timely and appropriate recapping of the telomere is possible, regulated uncapping of a telomere appears to be not only normal, but even required for optimal telomere maintenance. Telomere length and the presence of telomerase provide an example of a pair of interacting components that determine telomere capping function. Telomerase is dispensable in cells with sufficiently long telomeres; but in cells with short telomeres lacking telomerase, cells lose the ability to proliferate, and in some cell types, telomere fusions are increased. However, expressing telomerase can make even very short telomeres functional. Many interesting questions remain as to the mechanisms of these biological effects.

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

The ribonucleoprotein (RNP) enzyme telomerase from Saccharomyces cerevisiae adds telomeric DNA to chromosomal ends in short increments both in vivo and in vitro. Whether or not telomerase functions as a multimer has not been addressed previously. Here we show, first, that following polymerization, the telomerase RNP remains stably bound to its telomeric oligonucleotide reaction product. We then exploit this finding and a previously reported mutant telomerase RNA to demonstrate that, unexpectedly, the S. cerevisiae telomerase complex contains at least two functionally interacting RNA molecules that both act as templates for DNA polymerization. Here, functional telomerase contains at least two active sites.

The ribonucleoprotein enzyme telomerase adds telomeric DNA to chromosomal ends. In most eukaryotes the telomeric repeat units are repeated precisely, consistent with the action of a telomerase that faithfully copies its RNA template. In contrast, Saccharomyces cerevisiae telomeric repeats are degenerate, suggesting that its telomerase has unusual mechanistic properties. We mutated the S. cerevisiae telomerase RNA (TLC1) with a series of 3-base (GUG) substitutions in and next to the 17-nucleotide templating domain. All mutant telomerases were active in TLC1/tlc1 diploids and synthesized patterns of mixed wild-type and mutant telomeric repeats into telomeric DNA, consistent with nonprocessive action. Telomerase isolated from cells containing each mutated tlc1 allele by itself had altered reaction properties in vitro. One mutant template enzyme, 476GUG, was active in vivo and in vitro in the presence of wild-type TLC1 RNA but lacked detectable activity in its absence. Haploid tlc1-476GUG cells containing only this mutant tlc1 allele underwent senescence. Other tlc1 template region mutations allowed maintenance of shortened telomeres in vivo but altered specific enzymatic properties of telomerase in vitro, including induction of primer-template slippage (472GUG) or alteration of the 5' boundary of the template (467GUG). These data demonstrate that telomerase RNA bases influence enzyme activity profoundly, suggesting that their roles are not confined to serving simply as the template for this specialized reverse transcriptase.

Deletion of the telomerase RNA gene (TER1) in the yeast Kluyveromyces lactis results in gradual loss of telomeric repeats and progressively declining cell growth capability (growth senescence). We show that this initial growth senescence is characterized by abnormally large, defectively dividing cells and is delayed when cells initially contain elongated telomeres. However, cells that survive the initial catastrophic senescence emerge relatively frequently, and their subsequent growth without telomerase is surprisingly efficient. Survivors have lengthened telomeres, often much longer than wild type, but that are still subject to gradual shortening. Production of these postsenescence survivors is strongly dependent on the RAD52 gene. We propose that shortened, terminal telomeric repeat tracts become uncapped, promoting recombinational repair between them to regenerate lengthened telomeres in survivors. This process, which we term telomere cap-prevented recombination (CPR) may be a general alternative telomere maintenance pathway in eukaryotes.

The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA by copying a template sequence in the telomerase RNA. We studied the functional roles of specific residues in the Tetrahymena telomerase RNA template region. Unexpectedly, mutation of certain templating residues caused dramatic effects on specific aspects of the enzyme reaction, including loss of enzymatic fidelity and premature product dissociation. None of these fundamental changes in enzymatic action are explainable by altered base-pairing between the telomerase RNA and DNA substrate. These influences of specific template bases of the telomerase RNA on enzymatic properties of telomerase provide evidence for critical roles of these RNA residues in two active site functions--fidelity and DNA substrate/enzyme interaction.

The ciliated protozoan Tetrahymena thermophila contains two nuclei that differ dramatically in function, chromosome size and number, chromatin structure, and mode of division. It is possible that the telomeres of the two nuclei have different functions. Although macronuclear telomeric DNA has been well characterized and consists of tandem G4T2/C4A2 repeats that are synthesized by the enzyme telomerase, micronuclear telomeres have not been isolated previously. Here, we report the identification and cloning of micronuclear telomeres and demonstrate that although they contain the same terminal tandem G4T2 repeats as macronuclear telomeres, they are strikingly different in three respects. First, the tracts of G/C-rich telomeric repeats are approximately seven times longer in the micronucleus than in the macronucleus (approximately 2.0-3.4 vs. approximately 0.3-0.5 kb, respectively) from the same cell population. Second, the immediate telomere-associated sequences (TASs) from six different micronuclear chromosome ends have an unusually high G/C content and degree of homology to one another, unlike macronuclear TASs. The TAS from at least one micronuclear chromosome is unique to micronuclear telomeres and is not present in the macronucleus. Finally, and unexpectedly, all micronuclear telomere clones contain an inner homogeneous tract of a variant G4T3 repeat adjacent to the distal tract of G4T2 repeats. The native micronuclear telomeric DNA is composed of approximately 30% G4T3 repeats, corresponding to 0.6-1.0 kb per average telomere, positioned centromere-proximally to most or all of the G4T2 repeats. Neither the G4T3 sequence nor any other variant repeat is found in macronuclear telomeres. Furthermore, such a homogeneous tract of a variant repeat has not been found in the telomeres of any eukaryote.

During development of the somatic macronucleus of Tetrahymena thermophila, the rDNA is excised from its germ-line chromosome, rearranged into a palindrome, and amplified to 10(4) copies. We have identified a cis-acting germ-line mutation, rmm11/6, that prevents amplification of the rDNA in all but approximately 1 in 10(5) cells when it is the only rDNA allele in the developing macronucleus. The rmm11/6 mutation resides in a conserved element required for excision, the chromosome breakage sequence (Cbs) flanking the 3' end of the rDNA. Surprisingly, the rmm11/6 mutation only weakly affects excision of the rDNA from its germ-line location; at least 25% of cells heterozygous for this mutation correctly excise the affected rDNA allele. In heterozygotes, when this rDNA allele is excised, it is also poorly amplified. The rDNA amplification defect caused by this mutation is not overcome by delaying amplification with the DNA synthesis inhibitor aphidicolin, indicating that rDNA excision and amplification are not experimentally separable. Our experiments provide the first evidence that the capacity to amplify the rDNA is restricted in the developing macronucleus. We propose that the rmm11/6 mutation delays excision of the rDNA and that the developmental progression of the macronucleus past a restricted window for amplification is responsible for the severe amplification defect caused by this weak rDNA excision mutation.