Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo

The 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, in addition to providing a 16-nt template region for reverse transcription, has been proposed to act as a scaffold for protein subunits. Although accessory subunits of the telomerase ribonucleoprotein (RNP) complex function even when their b...

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Bibliographic Details
Published in:RNA (Cambridge) 2012-09, Vol.18 (9), p.1666-1678
Main Authors: Lebo, Kevin J, Zappulla, David C
Format: Article
Language:English
Subjects:
Base Sequence
DNA-Binding Proteins - metabolism
Models, Biological
Nucleic Acid Conformation
Protein Binding
Ribonucleoproteins - metabolism
RNA - chemistry
RNA - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - metabolism
Telomerase - chemistry
Telomerase - metabolism
Telomere - metabolism
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Summary:The 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, in addition to providing a 16-nt template region for reverse transcription, has been proposed to act as a scaffold for protein subunits. Although accessory subunits of the telomerase ribonucleoprotein (RNP) complex function even when their binding sites are relocated on the yeast telomerase RNA, the physical nature of the RNA scaffold has not been directly analyzed. Here we explore the structure-function organization of the yeast telomerase RNP by extensively stiffening the three long arms of TLC1, which connect essential and important accessory protein subunits Ku, Est1, and Sm(7), to its central catalytic hub. This 956-nt triple-stiff-arm TLC1 (TSA-T) reconstitutes active telomerase with TERT (Est2) in vitro. Furthermore, TSA-T functions in vivo, even maintaining longer telomeres than TLC1 on a per RNA basis. We also tested functional contributions of each stiffened arm within TSA-T and found that the stiffened Est1 and Ku arms contribute to telomere lengthening, while stiffening the terminal arm reduces telomere length and telomerase RNA abundance. The fact that yeast telomerase tolerates significant stiffening of its RNA subunit in vivo advances our understanding of the architectural and functional organization of this RNP and, more broadly, our conception of the world of lncRNPs.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.033555.112