Schweingruber, Christoph; Soffientini, Paolo; Ruepp, Marc-David; Bachi, Angela; Mühlemann, Oliver (2016). Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID). PLoS ONE, 11(3), e0150239. Public Library of Science 10.1371/journal.pone.0150239
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Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.
Item Type: |
Journal Article (Original Article) |
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Division/Institute: |
08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP) |
Graduate School: |
Graduate School for Cellular and Biomedical Sciences (GCB) |
UniBE Contributor: |
Schweingruber, Christoph, Ruepp, Marc-David, Mühlemann, Oliver |
Subjects: |
500 Science > 570 Life sciences; biology 500 Science > 540 Chemistry |
ISSN: |
1932-6203 |
Publisher: |
Public Library of Science |
Language: |
English |
Submitter: |
Christina Schüpbach |
Date Deposited: |
22 Mar 2016 11:07 |
Last Modified: |
05 Dec 2022 14:53 |
Publisher DOI: |
10.1371/journal.pone.0150239 |
PubMed ID: |
26934103 |
BORIS DOI: |
10.7892/boris.79234 |
URI: |
https://boris.unibe.ch/id/eprint/79234 |
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