Single-Molecule Imaging of Telomerase RNA Reveals a Recruitment-Retention Model for Telomere Elongation

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis. [Display omitted] •Live-cell and single-molecule imaging of telomerase RNA by MS2 tagging•Telomerase reverse transcriptase drives hTR exit from Cajal bodies•ATM (and ATR) facilitates hTR recruitment to telomeres•RNA-DNA base pairing promotes telomerase retention at telomeres Telomerase is the means by which many cancer cells achieve immortality. Laprade et al. developed an MS2-tagging approach to visualize single particles of hTR in cancer cells and reveal telomerase dynamics as hTR transits through Cajal bodies and is recruited and retained at telomeres.