The telomerase holoenzyme acts as a reverse transcriptase to elongate shortened, non-functional telomeres. This enzyme is composed of several protein subunits and assembled onto a scaffolding non-coding RNA, called Tlc1 in budding yeast. Like some other protein subunits (Est1, Est2, Est3, Pop1), Tlc1 is essential for telomerase activity and telomere maintenance in vivo. However, the pathways and mechanisms leading to an assembled and functional telomerase complex are not understood. Telomerase biogenesis has been studied via genetics and biochemistry, but real time in vivo data are by and large missing. As a readout for telomerase biogenesis, Tlc1 is a target of choice, as it is the limiting subunit for telomerase activity in Saccharomyces cerevisiae (its expression was measured at 25-30 RNAs/cell). Previous work showed that Tlc1 can shuttle between nucleus and cytoplasm, although no canonical ncRNA maturation is known to happen in the cytoplasm in yeast. Our lab previously used the MS2-GFP system to study Tlc1 dynamics and recruitment to telomeres in vivo, and the results revealed telomere-associated clusters of telomerase, called T-Recs, correlated with telomere elongation (Gallardo et al.; Mol Cell 2011).
In order to gain more detailed insights into Tlc1 ncRNA biogenesis, we are using a combination of the MS2-GFP and the Cre-Lox systems. The endogenous TLC1 locus was floxed so that the expressed Tlc1 untagged. The MS2 tag can be inserted at will via and inducible Cre-EBD protein. The new TLC1-MS2 locus gives rise to Tlc1-MS2 RNAs, which can be visualized via the MS2-GFP fusion protein. These newly transcribed, GFP-bound Tlc1 RNAs can thus be followed from the transcription site to the final target at telomeres by time-lapse microscopy in living cells. It is also possible to IP these RNAs in order to decipher how the telomerase complex assembles. This construct, named TLC1-MS2-IN, thus has the potential to allow studies on the first biogenesis steps of Tlc1 and telomerase. A similar construct, dubbed TLC1-MS2-OUT, allows for the inducible removal of the MS2 tag. This second construct allows studies of Tlc1 degradation, half-life, and telomerase nucleocytoplasmic recycling. Preliminary Southern- and Northern-blots results with this latter setup show that there is a lag between the genomic recombination and the differential RNA expression, suggesting that Tlc1 is a stable RNA that can last for the duration of at least one cell cycle.
This study, apart from giving insight into Tlc1 biogenesis and telomerase assembly, could thus help better define ncRNA maturation or degradation pathways in budding yeast.