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

PLoS One. 2016 Mar 2;11(3):e0150239. doi: 10.1371/journal.pone.0150239. eCollection 2016.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation
  • Carbon-Nitrogen Ligases / genetics
  • Carbon-Nitrogen Ligases / isolation & purification
  • Carbon-Nitrogen Ligases / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism*
  • Cell Line
  • Chromatography, Liquid
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification
  • Escherichia coli Proteins / metabolism
  • HeLa Cells
  • Humans
  • Immunoprecipitation / methods
  • Nonsense Mediated mRNA Decay*
  • Protein Interaction Mapping / methods*
  • Protein Interaction Maps*
  • RNA Helicases
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / isolation & purification
  • Repressor Proteins / metabolism
  • Tandem Mass Spectrometry
  • Trans-Activators / genetics
  • Trans-Activators / isolation & purification
  • Trans-Activators / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / isolation & purification
  • Transcription Factors / metabolism*


  • Carrier Proteins
  • Escherichia coli Proteins
  • RNA, Messenger
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • SMG5 protein, human
  • Trans-Activators
  • Transcription Factors
  • UPF2 protein, human
  • RNA Helicases
  • UPF1 protein, human
  • Carbon-Nitrogen Ligases
  • birA protein, E coli

Grants and funding

This work has been supported by the Swiss National Science Foundation (, grant 31003A_143717 to OM), by the canton of Bern (University intramural funding to OM), by the Holcim Stiftung zur Förderung der wissenschaftlichen Fortbildung (, to MDR), and by the Associazione Italiana per la Ricerca sul Cancro (, investigator grant 14578 to AB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.