Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies.

We dissected the importance of human telomerase biogenesis and trafficking pathways for telomere maintenance. Biological stability of human telomerase RNA (hTR) relies on H/ACA proteins, but other eukaryotes use other RNP assembly pathways. To investigate additional rationale for human telomerase assembly as H/ACA RNP, we developed a minimized cellular hTR. Remarkably, with only binding sites for telomerase reverse transcriptase (TERT), minimized hTR assembled biologically active enzyme. TERT overexpression was required for cellular interaction with minimized hTR, indicating that H/ACA RNP assembly enhances endogenous hTR-TERT interaction. Telomere maintenance by minimized telomerase was unaffected by the elimination of the telomerase holoenzyme Cajal body chaperone TCAB1 or the Cajal body scaffold protein Coilin. Surprisingly, wild-type hTR also maintained and elongated telomeres in TCAB1 or Coilin knockout cells, with distinct changes in telomerase action. Overall, we elucidate trafficking requirements for telomerase biogenesis and function and expand mechanisms by which altered telomere maintenance engenders human disease.

Human stem cell-based disease modeling: prospects and challenges.

Human stem cell-based disease models have great promise to advance our understanding of human disease. These models can be derived from patients with genetic disorders and manipulated with genome editing and myriad differentiation protocols to model pathologies in vitro. However, several challenges have impeded the full potential of stem cell-based in vitro disease modeling. Many genetically predisposed diseases take time to manifest and occur in specific tissue microenvironments, and these parameters are often not adequately modeled using conventional shorter-term monolayer cultures. These challenges must be overcome especially for cases where animal models also incompletely recapitulate the complex pathologies found in humans. As prominent ways to tackle these challenges we discuss here how advanced genome editing tools in human stem cells and human organoid cultures, specifically the example of intestinal organoids, contribute genetically defined models that recapitulate phenotypes of disease.

Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells.