Imagerie moléculaire de l’ARN de la télomérase dans des cellules cancéreuses humaines
Telomeres are shortened at each DNA replication cycle, due to the end replication problem. Their length dictates the proliferative capacity of eukaryotic cells. Too short telomeres, reaching the Hayflick limit, will lead the cells to replicative senescence. However, in stem cells and 90% of cancer cells telomere length is maintained by a ribonucleoproteic complex named telomerase. The assembly of this complex and its recruitment to telomeres are process under a fine regulation. Yet, how this regulation impacts the nuclear dynamics of telomerase is not fully understood. In this project, we developed a new method based on the MS2 system to image for the first time telomerase RNA (hTR) particles in living human cancer cells by fluorescence microscopy. The real time tracking of these particles at telomeres revealed that telomerase actively scan all telomeres by short interactions with the TPP1 shelterin protein. Long stable interaction needed for telomere elongation can be made by the pairing of hTR template and single stranded telomeric DNA under the control of the OB-fold domains of the shelterin protein POT1. By tracking telomerase particle during the cell cycle, we revealed that telomerase recruitment to telomeres is not restricted to S phase, when telomeres are elongated. By measuring the dynamics of hTR in Cajal bodies (CBs) with photo-activation and photo-bleaching technics we showed that hTERT play a role in the hTR exit from CBS. Finally, fluorescence intensity measures of hTR at telomeres showed the recruitment of only one particle on a telomere.
hTR hTERT single-molecule imaging Cajal Body in vivo RNA dynamics cancer