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Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID).
- Source :
-
PloS one [PLoS One] 2016 Mar 02; Vol. 11 (3), pp. e0150239. Date of Electronic Publication: 2016 Mar 02 (Print Publication: 2016). - Publication Year :
- 2016
-
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.
- Subjects :
- Biotinylation
Carbon-Nitrogen Ligases genetics
Carbon-Nitrogen Ligases isolation & purification
Carbon-Nitrogen Ligases metabolism
Carrier Proteins genetics
Carrier Proteins isolation & purification
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
RNA Helicases
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
Transcription Factors genetics
Transcription Factors isolation & purification
Carrier Proteins metabolism
Nonsense Mediated mRNA Decay
Protein Interaction Mapping methods
Protein Interaction Maps
RNA, Messenger metabolism
Trans-Activators metabolism
Transcription Factors metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 11
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 26934103
- Full Text :
- https://doi.org/10.1371/journal.pone.0150239