Biochemical characterization of the RNA helicase UPF1 involved in nonsense-mediated mRNA decay.
Degradation of eukaryotic mRNAs harboring a premature translation termination codon is ensured by the process of nonsense-mediated mRNA decay (NMD). The main effector of this quality-control pathway is the conserved RNA helicase UPF1 that forms a surveillance complex with the proteins UPF2 and UPF3. In all the organisms tested, the ATPase activity of UPF1 is essential for NMD. Here, we describe the expression of active recombinant UPF proteins and the reconstitution of the surveillance complex in vitro. To understand how UPF1 is regulated during NMD, we developed different biochemical approaches. We describe methods to monitor UPF1 binding to RNA, ATP hydrolysis and RNA unwinding in the presence of its binding partner UPF2. This functional analysis is an important complement for structural studies of protein complexes containing RNA helicases.
Institut de Biologie de l'Ecole Normale Supérieure, CNRS UMR8197, INSERM U1024, Paris Cedex 05, France.
Francesca
F
Fiorini
Fabien
F
Bonneau
Hervé
H
Le Hir
D006801:Humans; D059365:Nonsense Mediated mRNA Decay / Q000235:genetics / Q000502:physiology*; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D015534:Trans-Activators; D020365:RNA Helicases; C101828:UPF1 protein, human