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

Mol Cell. 2020 Jul 2;79(1):115-126.e6. doi: 10.1016/j.molcel.2020.05.005. Epub 2020 Jun 3.

Abstract

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.

Keywords: ATM; ATR; POT1; cancer cellsĆ; fluorescent in situ hybridization; hTR RNA; single-molecule imaging; telomerase; telomeres.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Coiled Bodies / metabolism*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism*
  • Gene Editing
  • HeLa Cells
  • Humans
  • Mutation
  • RNA / genetics
  • RNA / metabolism*
  • Single Molecule Imaging / methods*
  • Telomerase / genetics
  • Telomerase / metabolism*
  • Telomere / genetics
  • Telomere / metabolism*
  • Telomere Homeostasis*
  • Telomere-Binding Proteins / genetics
  • Telomere-Binding Proteins / metabolism

Substances

  • DNA, Single-Stranded
  • POT1 protein, human
  • Telomere-Binding Proteins
  • telomerase RNA
  • RNA
  • TERT protein, human
  • Telomerase