title: 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 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.|Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Schweingruber
authors.affiliation: IFOM-FIRC Institute of Molecular Oncology, Milan, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Soffientini
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Marc-David
authors.initials: MD
authors.lastname: Ruepp
authors.affiliation: IFOM-FIRC Institute of Molecular Oncology, Milan, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Bachi
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Oliver
authors.initials: O
authors.lastname: Mühlemann
mesh_terms: D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D059365:Nonsense Mediated mRNA Decay; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D020365:RNA Helicases; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C515801:SMG5 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C468286:UPF2 protein, human; D020365:RNA Helicases; C101828:UPF1 protein, human; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli