The RNA component of telomerase (telomerase RNA; TER) varies substantially both in sequence composition and size (from ;150 nucleotides [nt] to >1500 nt) across species. This dramatic divergence has hampered the identification of TER genes and a large-scale comparative analysis of TER sequences and structures among distantly related species. To identify by phylogenetic analysis conserved sequences and structural features of TER that are of general importance, it is essential to obtain TER sequences from evolutionarily distant groups of species, providing enough conservation within each group and enough variation among the groups. To this end, we identified TER genes in several yeast species with relatively large (>20 base pairs) and nonvariant telomeric repeats, mostly from the genus Candida. Interestingly, several of the TERs reported here are longer than all other yeast TERs known to date. Within these TERs, we predicted a pseudoknot containing U-AÁU base triples (conserved in vertebrates, budding yeasts, and ciliates) and a three-way junction element (conserved in vertebrates and budding yeasts). In addition, we identified a novel conserved sequence (CS2a) predicted to reside within an internal-loop structure, in all the budding yeast TERs examined. CS2a is located near the Est1p-binding bulge-stem previously identified in Saccharomyces cerevisiae. Mutational analyses in both budding yeasts S. cerevisiae and Kluyveromyces lactis demonstrate that CS2a is essential for in vivo telomerase function. The comparative and mutational analyses of conserved TER elements reported here provide novel insights into the structure and function of the telomerase ribonucleoprotein complex.

Summary Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain that is highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase and RNases P/MRP, from unrelated progenitor RNAs.

The telomerase, which is composed of both protein and RNA, maintains genome stability by replenishing telomeric repeats at the ends of chromosomes. Here, we use live-cell imaging to follow yeast telomerase RNA dynamics and recruitment to telomeres in single cells. Tracking of single telomerase particles revealed a diffusive behavior and transient association with telomeres in G1 and G2 phases of the cell cycle. Interestingly, concurrent with telomere elongation in late S phase, a subset of telomerase enzyme clusters and stably associates with few telomeres. Our data show that this clustering represents elongating telomerase and it depends on regulators of telomerase at telomeres (MRX, Tel1, Rif1/2, and Cdc13). Furthermore, the assay revealed premature telomere elongation in G1 in a rif1/2 strains, suggesting that Rif1/2 act as cell-cycle dependent negative regulators of telomerase. We propose that telomere elongation is organized around a local and transient accumulation of several telomerases on a few telomeres.

The assembly of a protective cap onto the telomeres of eukaryotic chromosomes suppresses genomic instability through inhibition of DNA repair activities that normally process accidental DNA breaks. We show here that the essential Cdc13-Stn1-Ten1 complex is entirely dispensable for telomere protection in non-dividing cells. However, Yku and Rap1 become crucially important for this function in these cells. After inactivation of Yku70 in G1-arrested cells, moderate but significant telomere degradation occurs. As the activity of cyclin-dependent kinases (CDK) promotes degradation, these results suggest that Yku stabilizes G1 telomeres by blocking the access of CDK1-independent nucleases to telomeres. The results indeed show that both Exo1 and the Mre11/Rad50/Xrs2 complex are required for telomeric resection after Yku loss in non-dividing cells. Unexpectedly, both asynchronously growing and quiescent G0 cells lacking Rap1 display readily detectable telomere degradation, suggesting an earlier unanticipated function for this protein in suppression of nuclease activities at telomeres. Together, our results show a high flexibility of the telomeric cap and suggest that distinct configurations may provide for efficient capping in dividing versus non-dividing cells.

In bakers' yeast, in vivo telomerase activity requires a ribonucleoprotein (RNP) complex with at least four associated proteins (Est2p, Est1p, Est3p, and Cdc13p) and one RNA species (Tlc1). The function of telomerase in maintaining chromosome ends, called telomeres, is tightly regulated and linked to the cell cycle. However, the mechanisms that regulate the expression of individual components of telomerase are poorly understood. Here we report that yeast RNase III (Rnt1p), a double-stranded RNA-specific endoribonuclease, regulates the expression of telomerase subunits and is required for maintaining normal telomere length. Deletion or inactivation of RNT1 induced the expression of Est1, Est2, Est3, and Tlc1 RNAs and increased telomerase activity, leading to elongation of telomeric repeat tracts. In silico analysis of the different RNAs coding for the telomerase subunits revealed a canonical Rnt1p cleavage site near the 3 end of Est1 mRNA. This predicted structure was cleaved by Rnt1p and its disruption abolished cleavage in vitro. Mutation of the Rnt1p cleavage signal in vivo impaired the cell cycle-dependent degradation of Est1 mRNA without affecting its steady-state level. These results reveal a new mechanism that influences telomeres length by controlling the expression of the telomerase subunits.The ends of eukaryotic chromosomes are capped with special structures made of tandem DNA repeats and associated proteins, called telomeres. These structures protect chromosomes from end-to-end fusion, recombination, and nucleolytic degradation (1, 2). However, because the conventional DNA replication machinery cannot fully duplicate the ends of linear chromosomes, telomeric DNA will shorten with each round of replication, leading to a replicative senescence (3). To solve this end replication problem, most eukaryotes use the activity of an enzyme called telomerase to ensure the maintenance of telomeric DNA (4). Telomerase is a ribonucleoprotein reverse transcriptase that can extend the 3Ј end of chromosomes using its RNA subunit as a template for the addition of telomeric repeats.In vitro, telomerase activity requires a reverse transcriptase (Tert, Est2p in yeast) and an RNA template (Terc, Tlc1 in yeast)