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 (coIP)
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.
R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This socalled
BioID method provides an alternative to the widely used co-immunoprecipitation (coIP)
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.
| Original language | English |
|---|---|
| Article number | e0150239 |
| Journal | PloS one |
| Volume | 11 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2 Mar 2016 |