A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs

Abstract

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.

Figure 1.

A common secondary structure model for budding yeast TERs reveals an element similar to the vertebrate CR4-CR5 domain. (A) A schematic representation of a common secondary structure for budding yeast TERs was drawn according to the secondary structure predictions for Kluyveromyces and Saccharomyces TERs shown in Figure S1. (B) A schematic representation of the vertebrate TER structure was drawn according to (41). Indicated are the various functional elements found in telomerase RNA. Secondary structure models for the S. cerevisiae (C), K. lactis (D), human (E), S. castelii (F), S. kluyveri (G) and C. serpentina (H) TWJs were predicted, as described in the Supplementary Data. Indicated in blue are nucleotides conserved within each group [in all six Kluyveromyces, all seven Saccharomyces sensu stricto or in at least 33 of 35 vertebrate TERs (>94%)]. In the S. castellii and S. kluyveri models, indicated in blue are nucleotides conserved with the rest of the Saccharomyces species. Indicated in orange are nucleotides conserved in at least 13 of 15 yeast TERs (>86%). Indicated in red are nine nucleotides conserved across yeast and vertebrates in at least 45 of the 50 TER sequences examined (>90%; see Figure S3). Circled are positions with covariations within the Kluyveromyces, Saccharomyces sensu stricto or the vertebrate groups, supporting the predicted structures. (I) A representation of the consensus TWJ structure for yeast and vertebrate TERs. Indicated in red are conserved base pairs and conserved nucleotides.

Figure 2.

The three-way junction is crucial for K. lactis telomerase function in vivo. The mutations introduced into the TWJ (A and B) and the resulting mean telomere length, as measured by Southern analysis (C), are summarized in (A). WT and mutant TER1 genes were introduced into K. lactis cells on a CEN-ARS plasmid, replacing the WT TER1 gene. These mutants contain an additional BclI template mutation that is incorporated into telomeres, introducing a BclI restriction site. Otherwise, the BclI mutation is phenotypically silent and can therefore be used to mark the nascent products of the investigated telomerase in vivo (see scheme in Figure S4). Genomic DNA was prepared from the sixth passage (∼90–120 generations), digested with EcoRI or EcoRI+BclI, electrophoresed on a 1% agarose gel, vacuum blotted onto a membrane, and hybridized first with a BclI-specific telomere probe (top) and then with a WT telomeric probe (bottom), as indicated on the right of (C). For simplicity, only a portion of the gel is shown, including 7 of the 12 K. lactis telomeres. (D) Typical colonies of the different strains taken at their forth passage. Impaired telomere maintenance is associated with rough colonies, as opposed to the smooth appearance of WT colonies.

Figure 3.

Stem 3 telomerase mutants are barely active in vitro. (A) A scheme showing the two telomerase substrates used, which are each 12 nt long, and which base pair to the telomerase template with their 3′-ends at positions 5 and 25 of the template [5(12) and 25(12), respectively]. A dashed arrow indicates the direction of polymerization, and the nucleotides added are shown above the arrow. (B) Partially purified telomerase fractions were prepared from WT, S3.1 and IL-U3toC3 strains. These fractions (10 μl each) were assayed for telomerase activity as described in Materials and Methods section. The nucleotides added by telomerase to the corresponding products are indicated on the sides of the panel, and the 10-mer oligonucleotide used as internal control is indicated by ‘IC’. (C) Slot-blot analysis with a TER1 probe reveals comparable amounts of TER in the fractions assayed for telomerase activity (10 μl each).

Figure 4.

Overexpression of Est2 and Est3 partially suppresses the CS5sub mutant phenotype. Genes encoding telomerase proteins were cloned into a high copy-number (2 μ) plasmid and introduced into the CS5sub mutant. Genomic DNA was prepared from the strains at their sixth passage, and telomere length and BclI-repeat incorporation were analyzed by Southern, as described in Figure 2 legend. Only WT hybridization is shown.