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.
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*
Substances
-
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 (
www.snf.ch, grant 31003A_143717 to OM), by the canton of Bern (University intramural funding to OM), by the Holcim Stiftung zur Förderung der wissenschaftlichen Fortbildung (
www.holcim-stiftung.ch, to MDR), and by the Associazione Italiana per la Ricerca sul Cancro (
www.airc.it, 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.