Human telomerase RNA-protein interactions.
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.
Bachand
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F
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada.
Triki
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I
Autexier
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C
D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D020134:Catalytic Domain; D002457:Cell Extracts; D002461:Cell Line, Transformed; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D006801:Humans; D020128:Inhibitory Concentration 50; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D002457:Cell Extracts; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; C492185:STAU1 protein, human; C117884:staufen protein, mammalian; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase