Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

Abstract

Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.

ACKNOWLEDGMENTS

This work was supported by NIH grant GM54198.

We thank Jacek Gaertig for strain CU522 and plasmid pBICH3, Jamie Williamson for MS2 coat protein plasmid, Bobby Hogg for purified TAP-MS2 coat protein, the Lemaux lab for generous use of their biolistic transformation system, and Martin Gorovsky and members of the Collins laboratory, including Keren Witkin and Catherine O'Connor, for advice and discussions on the manuscript.