Extraordinary diversity of telomeres, telomerase RNAs and their template regions in Saccharomycetaceae

Telomerase RNA (TR) carries the template for synthesis of telomere DNA and provides a scaffold for telomerase assembly. Fungal TRs are long and have been compared to higher eukaryotes, where they show considerable diversity within phylogenetically close groups. TRs of several Saccharomycetaceae were recently identified, however, many of these remained uncharacterised in the template region. Here we show that this is mainly due to high variability in telomere sequence. We predicted the telomere sequences using Tandem Repeats Finder and then we identified corresponding putative template regions in TR candidates. Remarkably long telomere units and the corresponding putative TRs were found in Tetrapisispora species. Notably, variable lengths of the annealing sequence of the template region (1–10 nt) were found. Consequently, species with the same telomere sequence may not harbour identical TR templates. Thus, TR sequence alone can be used to predict a template region and telomere sequence, but not to determine these exactly. A conserved feature of telomere sequences, tracts of adjacent Gs, led us to test the propensity of individual telomere sequences to form G4. The results show highly diverse values of G4-propensity, indicating the lack of ubiquitous conservation of this feature across Saccharomycetaceae.

Human-like telomeres in Zostera marina reveal a mode of transition from the plant to the human telomeric sequences

A previous study describing the genome of Zostera marina, the most widespread seagrass in the Northern hemisphere, revealed some genomic signatures of adaptation to the aquatic environment. Important features related to the ‘back-to-the-sea’ reverse evolutionary pathway were found, such as the loss of stomatal genes, while other functions like an algal-like cell wall composition were acquired. Beyond these, the genome structure and organization were comparable to the majority of plant genomes sequenced, except for one striking feature that went unnoticed at that time: the presence of human-like instead of the expected plant-type telomeric sequences. By using different experimental approaches including FISH, NGS and Ba131 analysis, we have confirmed its telomeric location in the chromosomes of Z. marina. We have also identified its telomerase RNA subunit (TR), confirming the presence of the human-type telomeric sequence in the template region. Remarkably, this region was found to be very variable even in clades with a highly conserved telomeric sequence across their species. Based on this observation, we propose that alternative annealing preferences in the template borders can explain the transition between the plant and human telomeric sequences. The further identification of paralogues of TR in several plant genomes brought us to the hypothesis that plants may keep an increased ability to change their telomeric sequence. We discuss the implications of this occurrence in the evolution of telomeres while introducing a mechanistic model for the transition from the plant to the human telomeric sequences.

Controlled Crystallization and Transformation of Carbonate Minerals with Dumbbell-like Morphologies on Bacterial Cell Templates

Research on the biogenic-specific polymorphism and morphology of carbonate has been gaining momentum in the fields of biomineralization and industrial engineering in recent years. We report the nucleation of carbonate particles on bacterial cell templates to produce a novel dumbbell-like morphology which was assembled by needle-like crystals of magnesium calcite or aragonite radiating out from both ends of the template bacterium. Mature dumbbell-like structures had a tendency to break apart in the central template region, which was made up mostly of weak amorphous carbonate. Further crystal growth, especially at the template region, transformed the broken pieces into spherulites. Rod-like cell templates were essential for the formation of dumbbell-like morphologies, and we propose a possible formation mechanism of the dumbbell-like morphology. Our findings provide new perspectives on the morphological formation mechanism in biomineralization systems and may have a potential significance in assembling composite materials suitable for industrial applications.

Mechanism of processive telomerase catalysis revealed by high-resolution optical tweezers

Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a newly developed single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. After release of multiple telomeric repeats from telomerase, we observed folding of product DNA into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.

Evidence for an Additional Base-Pairing Element between the Telomeric Repeat and the Telomerase RNA Template in Kluyveromyces lactis and Other Yeasts

ABSTRACT In all telomerases, the template region of the RNA subunit contains a region of telomere homology that is longer than the unit telomeric repeat. This allows a newly synthesized telomeric repeat to translocate back to the 3′ end of the template prior to a second round of telomeric repeat synthesis. In the yeast Kluyveromyces lactis, the telomerase RNA (Ter1) template has 30 nucleotides of perfect homology to the 25-bp telomeric repeat. Here we provide strong evidence that three additional nucleotides at positions −2 through −4 present on the 3′ side of the template form base-pairing interactions with telomeric DNA. Mutation of these bases can lead to opposite effects on telomere length depending on the sequence permutation of the template in a manner consistent with whether the mutation increases or decreases the base-pairing potential with the telomere. Additionally, mutations in the −2 and −3 positions that restore base-pairing potential can suppress corresponding sequence changes in the telomeric repeat. Finally, multiple other yeast species were found to also have telomerase RNAs that encode relatively long 7- to 10-nucleotide domains predicted to base pair, often with imperfect pairing, with telomeric DNA. We further demonstrate that K. lactis telomeric fragments produce banded patterns with a 25-bp periodicity. This indicates that K. lactis telomeres have preferred termination points within the 25-bp telomeric repeat.

Structural features of mouse telomerase RNA are responsible for the lower activity of mouse telomerase versus human telomerase

Human and mouse telomerases show a high degree of similarity in both the protein and RNA components. Human telomerase is more active and more processive than the mouse telomerase. There are two key differences between hTR [human TR (telomerase RNA)] and mTR (mouse TR) structures. First, the mouse telomerase contains only 2 nt upstream of its template region, whereas the human telomerase contains 45 nt. Secondly, the template region of human telomerase contains a 5-nt alignment domain, whereas that of mouse has only 2 nt. We hypothesize that these differences are responsible for the differential telomerase activities. Mutations were made in both the hTR and mTR, changing the template length and the length of the RNA upstream of the template, and telomerase was reconstituted in vitro using mouse telomerase reverse transcriptase generated by in vitro translation. We show that the sequences upstream of the template region, with a potential to form a double-stranded helix (the P1 helix) as in hTR, increase telomerase activity. The longer alignment domain increases telomerase activity only in the context of the P1 helix. Thus the TR contributes to regulating the level of activity of mammalian telomerases.

Stem-Loop IV of Tetrahymena Telomerase RNA Stimulates Processivity in trans

ABSTRACT Telomerase is a ribonucleoprotein enzyme responsible for the addition of telomeres onto the ends of chromosomes. Short or dysfunctional telomeres can lead to cell growth arrest, apoptosis, and genomic instability. Telomerase uses its RNA subunit to copy a short template region for telomere synthesis. To probe for regions of Tetrahymena telomerase RNA essential for function, we assayed 27 circularly permuted RNA deletions for telomerase in vitro activity and binding to the telomerase reverse transcriptase catalytic protein subunit. We found that stem-loop IV is required for wild-type telomerase activity in vitro and will stimulate processivity when added in trans.