Characterization of Phosphorylation- and RNA-Dependent UPF1 Interactors by Quantitative Proteomics

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.