Live Cell Imaging of Telomerase RNA Dynamics Reveals Cell Cycle-Dependent Clustering of Telomerase at Elongating Telomeres

Abstract

The telomerase, which is composed of both protein and RNA, maintains genome stability by replenishing telomeric repeats at the ends of chromosomes. Here, we use live-cell imaging to follow yeast telomerase RNA dynamics and recruitment to telomeres in single cells. Tracking of single telomerase particles revealed a diffusive behavior and transient association with telomeres in G1 and G2 phases of the cell cycle. Interestingly, concurrent with telomere elongation in late S phase, a subset of telomerase enzyme clusters and stably associates with few telomeres. Our data show that this clustering represents elongating telomerase and it depends on regulators of telomerase at telomeres (MRX, Tel1, Rif1/2, and Cdc13). Furthermore, the assay revealed premature telomere elongation in G1 in a rif1/2 strains, suggesting that Rif1/2 act as cell-cycle dependent negative regulators of telomerase. We propose that telomere elongation is organized around a local and transient accumulation of several telomerases on a few telomeres.

Replication of Telomeres and the Regulation of Telomerase

“…Disruption of the Tlc1 -Ku80 interaction reduces telomere length while not eliciting an est1 phenotype. When assessing telomere association of telomerase by live-cell imaging, it seemed that the Tlc1 -Ku80 interaction might only promote a transient interaction with telomeres, whereas the stable interaction in late S phase is mediated by Cdc13 -Est1 (Gallardo et al 2011). The Ku-mediated stimulation of telomerase may also involve its role in nuclear accumulation of Tlc1, which is reduced in Kudeficient cells (Gallardo et al 2008;Pfingsten et al 2012).…”

Telomerase caught in the act

“…The results presented reconcile the telomere/telomerase association models, providing an integrated view of how telomere homeostasis is achieved. 16 Live Cell Imaging of TLC1 RNA…”

“…[18][19][20] We recently adopted it to produce a version of TLC1 bearing ten MS2 stem loops near the 3' end of the mature RNA. 16 Co-expression of this tagged TLC1-10xMS2 RNA with the MS2 coat protein fused to GFP yielded in several fluorescent foci in yeast nuclei (named hereafter TLC1-GFP). FISH experiments directed against TLC1 RNA confirmed that the position of the TLC1-GFP fluorescent foci correspond to the absolute position of the TLC1 RNA.…”

“…Interestingly, while occurrence of T-Recs is restricted to late S phase in wild type cells, they can be detected in all the phases of the cell cycle in RIF deletion mutants. 16 Thus, mutations in RIF1 or RIF2 alleviate the cell cycle restriction of T-Rec accumulation. This cell cycle independent formation of T-Recs triggers unregulated telomerase activity, as rif1/2 cells blocked in G1 undergo telomerase-dependent elongation of a short telomere, unlike wild type cells.…”

“…This cell cycle independent formation of T-Recs triggers unregulated telomerase activity, as rif1/2 cells blocked in G1 undergo telomerase-dependent elongation of a short telomere, unlike wild type cells. 16 Therefore, the Rif1-Rif2 complex acts as a cell cycle specific inhibitor of telomerase activity by restricting T-Rec formation to the late S phase of the cell cycle. It is possible that in the absence of Rif proteins, telomere-bound MRX loads the Tel1 kinase and stimulates C-strand resection outside of S phase.…”