title: A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.
abstract: Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3' untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3' UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3' UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA; Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA;
authors.forename: Tatsuaki
authors.initials: T
authors.lastname: Kurosaki
authors.affiliation: Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA.
authors.forename: Wencheng
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA.
authors.forename: Mainul
authors.initials: M
authors.lastname: Hoque
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA; Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA;
authors.forename: Maximilian W-L
authors.initials: MW
authors.lastname: Popp
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA; Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA;
authors.forename: Dmitri N
authors.initials: DN
authors.lastname: Ermolenko
authors.affiliation: Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers New Jersey Medical School, Newark, New Jersey 07103, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Tian
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA; Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA; lynne_maquat@urmc.rochester.edu.
authors.forename: Lynne E
authors.initials: LE
authors.lastname: Maquat
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D054648:Protein Phosphatase 2 / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D020365:RNA Helicases / Q000378:metabolism; D020871:RNA Stability / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D059467:Transcriptome; D015854:Up-Regulation
chemical_list: D015534:Trans-Activators; D054648:Protein Phosphatase 2; D000251:Adenosine Triphosphatases; D020365:RNA Helicases; C101828:UPF1 protein, human
keywords: ATP-dependent RNA helicase; RIP-seq of transcriptome-binding sites for phospho-UPF1; UPF1; mRNP composition; nonsense-mediated mRNA decay; phosphorylation