Summary: Exon junction complexes (EJCs) loaded onto spliced mRNAs during splicing serve as molecular markers for various post-transcriptional gene-regulatory processes, including nonsense-mediated mRNA decay (NMD). Although the composition and structure of EJCs are well characterized, the mechanism regulating EJC deposition remains unknown. Here we find that threonine 163 (T163) within the RNA-binding motif of eIF4A3 (a core EJC component) is phosphorylated by cyclin-dependent protein kinases 1 and 2 in a cell cycle-dependent manner. T163 phosphorylation hinders binding of eIF4A3 to spliced mRNAs and other EJC components. Instead, it promotes association of eIF4A3 with CWC22, which guides eIF4A3 to an active spliceosome. These molecular events ensure the fidelity of specific deposition of the EJC ∼20–24 nt upstream of an exon-exon junction. Accordingly, NMD is affected by T163 phosphorylation. Collectively, our data provide evidence that T163 phosphorylation affects EJC formation and, consequently, NMD efficiency in a cell cycle-dependent manner. : Ryu et al. show that eIF4A3 (a core EJC component) is phosphorylated at the threonine 163 position by CDK1 and CDK2 in a cell cycle-dependent manner. This event triggers EJC remodeling and affects NMD efficiency in a cell cycle-dependent manner. Keywords: exon junction complex, eIF4A3, nonsense-mediated mRNA decay, CDK, phosphorylation, cell cycle
