RNA



METHODS

Expression of hTERT and hTR in cis reconstitutes an active human telomerase ribonucleoprotein


FRANÇOIS  BACHAND a1a2, GEORGE  KUKOLJ a3p1 and CHANTAL  AUTEXIER a1a2c1
a1 Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, H3A 2B2 Canada
a2 Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis, Jewish General Hospital, Montréal, Québec, H3T 1E2 Canada
a3 Department of Microbiology and Immunology, McGill University, Montréal, Québec, H3A 2B4 Canada

Abstract

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3′ end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

(Received February 7 2000)
(Revised March 9 2000)
(Accepted March 17 2000)


Key Words: cis expression; rabbit reticulocyte lysate; reverse transcriptase; RNP reconstitution; S. cerevisiae.

Correspondence:
c1 Reprint requests to: Chantal Autexier, Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis, Jewish General Hospital, Montréal, Québec, H3T 1E2 Canada.
p1 Present address: Boehringer Ingelheim Canada Ltd., BioMéga Research Division, Laval, Québec, H7S 2G5 Canada.