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Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID).

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Schweingruber Christoph, Soffientini Paolo, Ruepp Marc-David, Bachi Angela, Mühlemann Oliver,
Project Quality control of gene expression: towards understanding mechanism and physiological role of nonsense-mediated mRNA decay (NMD)
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Original article (peer-reviewed)

Journal PloS one
Volume (Issue) 11(3)
Page(s) 0150239 - 0150239
Title of proceedings PloS one
DOI 10.1371/journal.pone.0150239

Open Access

URL http://boris.unibe.ch/79234/
Type of Open Access Repository (Green Open Access)

Abstract

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.
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