Exploring TERRA (TElomeric Repeat-containing RNA) Expression and Regulation During Cell Growth in Saccharomyces cerevisiae
The physical ends of eukaryotic chromosomes consist of repetitive DNA sequences, which are associated with specialized proteins forming a nucleoprotein structure essential for the integrity of the linear chromosomes, and are known as telomeres. Telomerase is an enzyme responsible for the maintenance of  the telomeric repeats at the end of the chromosomes. Telomerase is a ribonucleoprotein, which contains a catalytic subunit that possesses reverse transcriptase activity, and a RNA subunit that acts as a template, since it possess the telomeric repeat sequences necessary to amplify telomere ends. Telomeres are transcribed in most eukaryotes into a non-coding RNA know as TERRA (Telomeric repeats-containing RNA). It has been proposed that TERRA may act as a regulator of telomere homeostasis, and as an inhibitor of telomerase, however, its specific function is still unknown. In Saccharomyces cerevisiae, TERRA is rapidly degraded by the 5’-3’ Rat1 exonuclease, which has hampered its study by classic biochemical experiments in yeast.   In this thesis, we report the use of cytological approaches to study TERRA in budding yeast. Two different approaches were used for this purpose: the fluorescent in-situ hybridization (FISH) and the labeling of TERRA by the MS2-GFP system, which allow the visualization of TERRA transcripts form a single telomere in living cells. With these two approaches, we observed that TERRA is expressed from a single telomere and accumulates as a single perinuclear foci, in a small percentage of cells population. We also demonstrate that TERRA expression occurs due to telomere shortening.  We demonstrate that TERRA interacts in vivo with the telomerase RNA (TLC1) in yeast. Telomere elongation depends on the action of several telomerase molecules that are visible as clusters, which associate with telomeres in late S phase in yeast, and mammalian cells. In adidition, we show that TERRA stimulates the nucleation of telomerase clusters. By performing time course experiments of TERRA and TLC1 RNA in live cells, we observed that TERRA acts as a scaffold for generating telomerase clusters, which are then recruited in late S phase to the telomere from which TERRA molecules originated. The recruitment of TERRA to its telomere of origin is dependent on factors that control telomerase recruitment at telomeres like: Mre11, Tel1 and the yKu complex. We propose that a short telomere expresses TERRA to assemble and organize telomerase molecules, which later on allows their recruitment at the short telomere, where elongation is needed.  Finally we showed an up-regulation of TERRA, and telomerase RNA TLC1, accompanied by a predominant cytoplasmic localization as cell growth progresses from exponential growth to diauxic shift, and stationary phase. In these conditions, TERRA foci co-localize with TLC1 RNA foci, suggesting that the function of TERRA as a scaffold molecule to generate telomerase cluster is necessary for this yeast cell growth phases.
Telomeric repeats-containing RNA (TERRA) Telomere Telomerase Saccharomyces cerevisiae MS2-GFP Non-coding RNA Microscopy Live cell imaging Cellular localization Logarithmic growth Diauxic shift Stationary phase