Identification of interactions in the NMD complex using proximity-dependent biotinylation (bioid)

Abstract

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA∗) allows stringent streptavidin affinity purification of proximal proteins. This socalled 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 posttranscriptional 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.