RESUMEN
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.
Asunto(s)
Cuerpos Enrollados/metabolismo , ADN de Cadena Simple/metabolismo , ARN/metabolismo , Imagen Individual de Molécula/métodos , Telomerasa/metabolismo , Homeostasis del Telómero , Telómero/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN de Cadena Simple/genética , Edición Génica , Células HeLa , Humanos , Mutación , ARN/genética , Complejo Shelterina , Telomerasa/genética , Telómero/genética , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Pluripotency is at the crossroads of stem cell research and biology of reproduction. The mature metaphase II oocyte contains the key factors for pluripotency induction and maintenance as assessed by its capacity to reprogram somatic nuclei. The cumulus cells (CCs) niche that surrounds the oocyte is crucial for its maturation and presumably for the oocyte to acquire its competence to confer pluripotency. In this study, we examined whether cells cultured from the human mature metaphase II oocyte CC niche (hCC) could be used as feeders for the propagation of human induced pluripotent stem cells. The induced pluripotent (iPS) cells cultured on hCC (hCC-iPS) were assessed for their pluripotency potential by their expression of pluripotency-associated genes such as Oct4, Nanog, and TRA1-60 and their competence to differentiate into the three germ layers in vitro (embryoid bodies) as well as in vivo (teratoma formation). We show that not only the hCC-iPS cells maintained their pluripotency potential, but they also exhibited much better self-renewal performance in terms of proliferation rate compared to the same cells cultured on human foreskin fibroblast (hFF) feeders (hFF-iPS). A comparative gene expression profile study of hCC and hFF revealed significant differences (P < 0.05) in expression of cellular matrix components and an upregulation in hCC of genes known to be important players in cell proliferation such as interleukin 6 gene (IL6).