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.
Highlights
The understanding of cellular mechanisms at the molecular level requires the elucidation of protein-protein interactions in vivo
The enzymatic trans-biotinylation by BirAÃ permanently marks proteins before lysis in intact cells and because it depends on two substrates that are only available in small amounts in the lysate, biotinylation after lysis should not occur in BioID
Consistent with this expectation, we could not detect any biotinylation occuring in the lysate under our assay conditions, neither when biotin was directly added to the lysate (Fig 1B) nor when it was supplemented from other cell lysates (Fig 1C)
Summary
The understanding of cellular mechanisms at the molecular level requires the elucidation of protein-protein interactions in vivo. For large multi-factor complexes assembled on mRNAs, co-immunoprecipitation (co-IP) assays often identify many interactors that are only peripheral components and complicate the interpretation of such results with the risk that the plethora of apparent interactors might conceal truly insightful mechanistic connections. The co-IP of UPF1, a central factor in the nonsense-mediated mRNA decay (NMD) pathway, followed by explorative mass spectrometry previously revealed a large number of PLOS ONE | DOI:10.1371/journal.pone.0150239. Probing NMD Factors by BioID and analysis, decision to publish, or preparation of the manuscript The co-IP of UPF1, a central factor in the nonsense-mediated mRNA decay (NMD) pathway, followed by explorative mass spectrometry previously revealed a large number of PLOS ONE | DOI:10.1371/journal.pone.0150239 March 2, 2016
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