Live-cell imaging of budding yeast telomerase RNA and TERRA

• Telomerase dynamics can be studied using live cell imaging of its RNA subunit.
• Live cell imaging allows observation of telomerase RNA single molecules and clusters.
• Expression of TERRA from a single telomere can be detected by live cell imaging.
• Single cell imaging allows detection of transient expression of TERRA.

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs.