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

Schweingruber, Christoph; Soffientini, Paolo; Ruepp, Marc-David; Bachi, Angela; Mühlemann, Oliver (2016). Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID). PLoS ONE, 11(3), e0150239. Public Library of Science 10.1371/journal.pone.0150239

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Schweingruber, Christoph; Ruepp, Marc-David and Mühlemann, Oliver

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

1932-6203

Publisher:

Public Library of Science

Language:

English

Submitter:

Christina Schüpbach

Date Deposited:

22 Mar 2016 11:07

Last Modified:

22 Mar 2016 11:07

Publisher DOI:

10.1371/journal.pone.0150239

PubMed ID:

26934103

BORIS DOI:

10.7892/boris.79234

URI:

https://boris.unibe.ch/id/eprint/79234

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