Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo
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The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.
KeywordsTelomerase TAP-MS AtPOT1a Pontin Reptin PURα1
We thank Dr. David Potěšil, Prof. Jiří Fajkus and Assoc. Prof. Zbyněk Zdráhal (CEITEC, Masaryk University Brno, Czech Republic) and members of the research group Molecular Complexes of Chromatin and Proteomics Core Facility for fruitful discussions during past years of the project.
Compliance with ethical standards
This work was supported by the Grant Agency of the Czech Republic (13-06943S to ES), by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601), by the Academy of Sciences of the Czech Republic (RVO 68081707) and Masaryk University (MUNI/C/0981/2010 to JM). The MS analyses were carried out with support of the Proteomics Core Facility of CEITEC.
Conflict of interest
The authors declare that they have no conflict of interest.
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