Expression of hTERT and hTR in cis reconstitutes and active human telomerase ribonucleoprotein.
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.
Bachand
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Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada.
Kukolj
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Autexier
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C
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D011994:Recombinant Proteins; C422952:telomerase RNA; D012313:RNA; C509186:TERT protein, human; D019098:Telomerase