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Nucleic Acids Research 2005 33(17):5446-5457; doi:10.1093/nar/gki848
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Published online 28 September 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

Delphine T. Marie-Egyptienne1,4, Maria Antonietta Cerone1,4, J. Arturo Londoño-Vallejo2 and Chantal Autexier1,3,4,*

1Department of Anatomy and Cell Biology, Institut Curie 26 rue d'Ulm, 75248 Paris, CEDEX 05, France 2UMR7147—Pavillon Trouillet-Rossignol, Institut Curie 26 rue d'Ulm, 75248 Paris, CEDEX 05, France 3Department of Medicine, McGill University Montréal, Québec, Canada H3A 2B2 4Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital Montréal, Québec, Canada H3T 1E2

*To whom correspondence should be addressed. Tel: +1 514 340 8260; Fax: +1 514 340 8295; Email: chantal.autexier{at}mcgill.ca

Received April 21, 2005. Revised July 22, 2005. Accepted September 1, 2005.

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.


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