Targeted 2′-O Methylation at a Nucleotide within the Pseudoknot of Telomerase RNA Reduces Telomerase Activity In Vivo

Abstract

Telomerase RNA is an essential component of telomerase, a ribonucleoprotein enzyme that maintains chromosome ends in most eukaryotes. Here we employ a novel approach, namely, RNA-guided RNA modification, to assess whether introducing 2′-O methylation into telomerase RNA can influence telomerase activity in vivo. We generate specific 2′-O methylation sites in and adjacent to the triple helix (within the conserved pseudoknot structure) of Saccharomyces cerevisiae telomerase RNA (TLC1). We show that 2′-O methylation at U809 reduces telomerase activity, resulting in telomere shortening, whereas 2′-O methylation at A804 or A805 leads to moderate telomere lengthening. Importantly, we also show that targeted 2′-O methylation does not affect TLC1 levels and that 2′-O-methylated TLC1 appears to be efficiently assembled into telomerase ribonucleoprotein. Our results demonstrate that RNA-guided RNA modification is a highly useful approach for modulating telomerase activity.

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Articles of Significant Interest Selected from This Issue by the Editors

We thank T. Cech, K. Friedman, and K. Goodrich (University of Colorado) for their generous gift of the YKF103 strain and X. Bi and H. Kuzmiak (University of Rochester, Department of Biology) for the yHK53 strain. We also thank the members (J. Karijolich, in particular) of the Yu laboratory for valuable discussions.

This work was supported by grant GM62937 (to Y.-T.Y.) from the National Institutes of Health.