A second essential function of the Est1-binding arm of yeast telomerase RNA

The enzymatic ribonucleoprotein telomerase maintains telomeres in many eukaryotes, including humans, and plays a central role in aging and cancer. Saccharomyces cerevisiae telomerase RNA, TLC1, is a flexible scaffold that tethers telomerase holoenzyme protein subunits to the complex. Here we test th...

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Bibliographic Details
Published in:RNA (Cambridge) 2015-05, Vol.21 (5), p.862-876
Main Authors: Lebo, Kevin J, Niederer, Rachel O, Zappulla, David C
Format: Article
Language:English
Subjects:
Base Sequence
Conserved Sequence
Molecular Sequence Data
Mutation
Nucleic Acid Conformation
Organisms, Genetically Modified
Protein Binding
Ribonucleoproteins - chemistry
Ribonucleoproteins - genetics
Ribonucleoproteins - physiology
RNA - chemistry
RNA - genetics
RNA - physiology
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - physiology
Telomerase - chemistry
Telomerase - genetics
Telomerase - physiology
Telomere - metabolism
Telomere Homeostasis - genetics
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Summary:The enzymatic ribonucleoprotein telomerase maintains telomeres in many eukaryotes, including humans, and plays a central role in aging and cancer. Saccharomyces cerevisiae telomerase RNA, TLC1, is a flexible scaffold that tethers telomerase holoenzyme protein subunits to the complex. Here we test the hypothesis that a lengthy conserved region of the Est1-binding TLC1 arm contributes more than simply Est1-binding function. We separated Est1 binding from potential other functions by tethering TLC1 to Est1 via a heterologous RNA-protein binding module. We find that Est1-tethering rescues in vivo function of telomerase RNA alleles missing nucleotides specifically required for Est1 binding, but not those missing the entire conserved region. Notably, however, telomerase function is restored for this condition by expressing the arm of TLC1 in trans. Mutational analysis shows that the Second Essential Est1-arm Domain (SEED) maps to an internal loop of the arm, which SHAPE chemical mapping and 3D modeling suggest could be regulated by conformational change. Finally, we find that the SEED has an essential, Est1-independent role in telomerase function after telomerase recruitment to the telomere. The SEED may be required for establishing telomere extendibility or promoting telomerase RNP holoenzyme activity.
ISSN:1355-8382
1469-9001
DOI:10.1261/rna.049379.114