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.
Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A ⅐ U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C ⅐ C ؉ triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C ⅐ U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerasespecific function.Telomerase, a ribonucleoprotein reverse transcriptase, makes up for losses caused by incomplete DNA replication and degradation, by adding species-specific, 5-to 26-nucleotide (nt) repeats onto the telomere termini (reviewed in reference 2). The telomerase complex contains an RNA subunit (TER) (TLC1 in Saccharomyces cerevisiae), a catalytic reverse transcriptase (TERT) (Est2 in S. cerevisiae), and several other protein components. Unlike other reverse transcriptases, telomerase specializes in repeatedly copying a short RNA template within its integral RNA component.TERs are highly divergent, being conserved in sequence only among closely related species. Phylogenetic covariation was used to predict conserved secondary structures for evolutionarily close species of ciliates (14, 24), vertebrates (4), Kluyveromyces budding yeasts of the K. marxianus cluster (31, 32), and Saccharomyces sensu stricto (3,7,13,37). Limited similarity in the general architecture was observed among these models, consisting of three long arms and a catalytic core domain (4, 13). Although nontemplate regions are essential for the assembly, regulation, and function of telomerase, their specific roles are still unclear (reviewed in reference 30). We hypothesized that important functional elements would exhibit better conservation in their tertiary structures, rather than in their secondary structures or sequences. Solving these tertiary structures may provide insights into their conserved functions.Pseudoknot elements were found to be critical for telomerase function in ciliates (28), vertebrates (4), and Kluyveromyces (32). For S. cerevisiae, alternative pseud...
The telomerase ribonucleoprotein copies a short template within its integral RNA moiety onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Non-template regions of telomerase RNA (TER) are also crucial for telomerase function, yet they are highly divergent in sequence among species and their roles are largely unclear. Using both phylogenetic and mutational analyses, we predicted secondary structures for TERs from Kluyveromyces budding yeast species. A comparison of these secondary structure models with the published model for the Saccharomyces cerevisiae TER reveals a common arrangement into three long arms, a templating domain in the center and several conserved elements in the same positions within the structure. One of them, a three-way junction element, is highly conserved in budding yeast TERs. This element also shows sequence and structure similarity to the critical CR4-CR5 activating domain of vertebrate TERs. Mutational analysis in Kluyveromyces lactis confirmed that this element, and in particular the residues conserved across yeast and vertebrates, is critical for telomerase action both in vivo and in vitro. These findings demonstrate that despite the extreme divergence of TER sequences from different organisms, they do share conserved elements, which presumably carry out common roles in telomerase function.
Telomerase is a ribonucleoprotein complex that extends the 3′ ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C + •G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo.RNA triplex | yeast | RNA structure | Hoogsteen T elomerase is a ribonucleoprotein complex that extends the 3′ ends of eukaryotic chromosomes by adding successive telomere DNA repeats using an internal RNA template and a specialized reverse transcriptase (1, 2). Telomeres are the protein-DNA complexes that form the ends of linear chromosomes and protect them from end-to-end fusion and degradation (3, 4). Telomerase is of significant medical interest owing to the correlation between telomere length and human health and the association of telomerase activity with cancer (5, 6). In the absence of telomerase activity, telomeres shorten with each round of cell division because of exonuclease digestion and the inability of conventional DNA polymerases to fully replicate linear chromosomes. Shortening past a critical length leads to cell cycle arrest and/or apoptosis (7). Telomerase activity is undetectable in most somatic cells, resulting in telomere attrition with each cell cycle (8, 9). On the other hand, telomerase is active in, and essential for the proliferation of, the germ line, some epithelial, haemopoietic, and stem cells, as well as ∼90% of cancer cell lines (10, 11). A number of inherited diseases are associated with telomere shortening due to telomerase insufficiency, such as dyskeratosis congenita, aplastic anemia, and pulmonary fibrosis (12-15).The telomerase holoenzyme consists of the telomerase reverse transcriptase (TERT) and telomerase RNA (TER), which are essential and sufficient for catalytic activity in vitro (16), and several species-specific accessory proteins. TERs are highly divergent in size and sequence between species, ranging from ∼150 nt in ciliates, ∼450 nt in vertebrates, to more than 2,000 nt in some fungi (17). TERs provide the template for telomeric DNA synthesis but also contain other domains that are essential for telomerase ass...
The ribonucleoprotein telomerase contains two essential components: telomerase RNA (TER) and telomerase reverse transcriptase (TERT, Est2 in yeast). A small portion of TER, termed the template, is copied by TERT onto the chromosome ends, thus compensating for sequence loss due to incomplete DNA replication and nuclease action. Although telomerase RNA is highly divergent in sequence and length across fungi and mammals, structural motifs essential for telomerase function are conserved. Here, we show that Est2 from the budding yeast Kluyveromyces lactis (klEst2) binds specifically to an essential three-way junction (TWJ) structure in K. lactis TER, which shares a conserved structure and sequence features with the essential CR4-CR5 domain of vertebrate telomerase RNA. klEst2 also binds specifically to the template domain, independently and mutually exclusive of its interaction with TWJ. Furthermore, we present the high-resolution structure of the klEst2 telomerase RNA-binding domain (klTRBD). Mutations introduced in vivo in klTRBD based on the solved structure or in TWJ based on its predicted RNA structure caused severe telomere shortening. These results demonstrate the conservation and importance of these domains and the multiple protein–RNA interactions between Est2 and TER for telomerase function.
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