A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation.
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.
Marie-Egyptienne
Delphine T
DT
Department of Anatomy and Cell Biology, Institut Curie 26 rue d'Ulm, 75248 Paris, CEDEX 05, France.
Cerone
Maria Antonietta
MA
Londoño-Vallejo
J Arturo
JA
Autexier
Chantal
C
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*; D013768:Tetrahymena / Q000201:enzymology / Q000235:genetics*
D004268:DNA-Binding Proteins; C422952:telomerase RNA; D012313:RNA; D019098:Telomerase