Quantitative proteomics of affinity purified yeast NMD complexes
ABSTRACT: Nonsense-mediated mRNA decay (NMD) is a major translation-dependent RNA degradation pathway required for embryo development and telomere maintenance. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals but a model of the NMD machinery compatible with all eukaryotes is not yet available. We performed the first large-scale quantitative characterization of yeast NMD complexes through affinity purification and mass-spectrometry with 7 different NMD-related factors, with or without Rnase, in strains deleted or not for NMD genes. This extensive characterization of NMD complexes identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained, in addition to Upf1, the mRNA decapping enzyme and two potential equivalents of mammalian Smg6/5/7: Nmd4 and Ebs1. Like the Smg proteins, Nmd4 and Ebs1 were required for efficient NMD. Our results suggest that the core eukaryotic NMD machinery is conserved across species and operates through successive Upf1-bound Detector and Effector complexes.