Nucleic Acids Research, 2001, Vol. 29, No. 16 3385-3393
© 2001 Oxford University Press
Human telomerase RNA–protein interactions
1Department of Anatomy and Cell Biology, McGill University, Montréal, Québec H3A 2B2, Canada and 2Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis, Jewish General Hospital, Montréal, Québec H3T 1E2, Canada
Telomere length is maintained in most eukaryotic cells by telomerase. The core components of this ribonucleoprotein (RNP) enzyme include a protein catalytic subunit, composed of motifs conserved among reverse transcriptases (RT), and an RNA subunit that contains a short template sequence essential for the synthesis of telomeric repeats. We developed an electrophoretic mobility shift assay using active telomerase partially purified from 293 cells and radiolabeled, in vitro-transcribed human telomerase RNA (hTR) to investigate the molecular interactions of the human telomerase RT (hTERT) and telomerase-associated proteins with hTR. A specific hTR–protein complex was identified and shown to contain hTERT and human Staufen by antibody supershift assays. Variants of hTR altered in distinct structural elements were analyzed for their ability to competitively inhibit complex formation. Human telomerase RNAs lacking the CR4-CR5 domain were poor inhibitors of hTR–protein complex formation, suggesting that the CR4-CR5 domain of hTR is a potential protein-binding site. Furthermore, alterations in the telomerase RNA pseudoknot’s P3 helix, the CR7 domain, or the H/ACA box efficiently inhibited formation of the complex, indicating that these domains are dispensable for the assembly of a telomerase RNP in vitro. Potential telomerase-associated proteins that bind hTR were also identified using a UV cross-linking assay.
* To whom correspondence should be addressed at: Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis, Jewish General Hospital, 3755 Côte Ste-Catherine Road, Montréal, Québec H3T 1E2, Canada. Tel: +1 514 340 8260; Fax: +1 514 340 8295; Email: cautex{at}po-box.mcgill.ca The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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