Characterization of phosphorylation- and RNA-dependent UPF1 interactors by quantitative proteomics

J Proteome Res. 2014 Jun 6;13(6):3038-53. doi: 10.1021/pr5002143. Epub 2014 May 6.


Human up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase and phosphoprotein implicated in several biological processes but is best known for its key function in nonsense-mediated mRNA decay (NMD). Here we employed a combination of stable isotope labeling of amino acids in cell culture experiments to determine by quantitative proteomics UPF1 interactors. We used this approach to distinguish between RNA-mediated and protein-mediated UPF1 interactors and to determine proteins that preferentially bind the hypo- or the hyper-phosphorylated form of UPF1. Confirming and expanding previous studies, we identified the eukaryotic initiation factor 3 (eIF3) as a prominent protein-mediated interactor of UPF1. However, unlike previously reported, eIF3 binds to UPF1 independently of UPF1's phosphorylation state. Furthermore, our data revealed many nucleus-associated RNA-binding proteins that preferentially associate with hyper-phosphorylated UPF1 in an RNase-sensitive manner, suggesting that UPF1 gets recruited to mRNA and becomes phosphorylated before being exported to the cytoplasm as part of the mRNP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Phosphorylation
  • Protein Interaction Mapping
  • Protein Interaction Maps
  • Protein Processing, Post-Translational
  • Proteomics
  • RNA Helicases
  • RNA, Messenger / metabolism*
  • Trans-Activators / metabolism*


  • RNA, Messenger
  • Trans-Activators
  • RNA Helicases
  • UPF1 protein, human