The ribonucleoprotein enzyme telomerase synthesizes DNA at the ends of chromosomes. Although the telomerase catalytic protein subunit (TERT) is well conserved, the RNA component is rapidly evolving in both size and sequence. Here, we reduce the 1,157-nucleotide (nt) Saccharomyces cerevisiae TLC1 RNA to a size smaller than the 451-nt human RNA while retaining function in vivo. We conclude that long protein-binding arms are not essential for the RNA to serve its scaffolding function. Although viable, cells expressing Mini-T have shortened telomeres and reduced fitness as compared to wild-type cells, suggesting why the larger RNA has evolved. Previous attempts to reconstitute telomerase activity in vitro using TLC1 and yeast TERT (Est2p) have been unsuccessful. We find that substitution of Mini-T for wild-type TLC1 in a reconstituted system yields robust activity, allowing the contributions of individual yeast telomerase components to be directly assessed.
*Note: In the supplementary information initially published online to accompany this article, the secondary structure model for a 436-nt mini-T is shown in Supplementary Figure 1, but the legend describes a 500-nt mini-T model. The secondary structure model for the 500-nt mini-T has now been supplied by the authors. The error has been corrected online.
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We thank A.J. Zaug (University of Colorado, Boulder, Colorado, USA) for plasmid T7-ProA-Est2p and A.G. Seto (present affiliation: Harvard Medical School, Cambridge, Massachusetts, USA) for telomerase immunopurified from yeast. Thanks also to A.J. Zaug for performing the telomerase activity assays that included human TERT. This research was supported in part by grant GM28039 from the US National Institutes of Health.
The authors declare no competing financial interests.
Mfold secondary structure model of Mini-T(500). (PDF 907 kb)
Mini-T(500) RNA accumulation when expressed from the chromosome. (PDF 355 kb)
Mini-T RNA levels during log phase cell growth. (PDF 734 kb)
Mini-T(64) and (67) do not yield reconstituted activity. (PDF 586 kb)
Reconstituted core mini-telomerase has activity very similar to wild-type telomerase from yeast. (PDF 1039 kb)
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Zappulla, D., Goodrich, K. & Cech, T. A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nat Struct Mol Biol 12, 1072–1077 (2005). https://doi.org/10.1038/nsmb1019
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