NMD factors UPF2 and UPF3 bridge UPF1 to the exon junction complex and stimulate its RNA helicase activity

Abstract

Nonsense-mediated mRNA decay (NMD) eliminates mRNAs containing a premature translation termination codon through the recruitment of the conserved NMD factors UPF1, UPF2 and UPF3. In humans, a dynamic assembly pathway allows UPF1 to join UPF2 and UPF3 recruited to the mRNA by the exon-junction complex (EJC). Here we show that the recombinant EJC core is sufficient to reconstitute, with the three UPF proteins, a stable heptameric complex on RNA. The EJC proteins MAGOH, Y14 and eIF4AIII provide a composite binding site for UPF3b that serves as a bridge to UPF2 and UPF1. In the UPF trimeric complex, UPF2 and UPF3b cooperatively stimulate both ATPase and RNA helicase activities of UPF1. This work demonstrates that the EJC core is sufficient to stably anchor the UPF proteins to mRNA and provides insights into the regulation of its central effector, UPF1.

SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs

“…The latter is in agreement with data published during the course of our studies demonstrating that UPF2 binds UPF1 residues 123-213 (Kadlec et al 2006). It makes sense that STAU1 would bind the cysteine-and histidinerich CH domain of UPF1 as does UPF2 in view of a recent report that the CH domain serves as an intramolecular regulator of UPF1 activities: Just as UPF2:UPF3X binding to the CH domain of UPF1 has been proposed to prevent the cis-inhibitory effect of the domain so as to stimulate UPF1 RNA-dependent ATPase and 59-to-39 helicase activities (Chamieh et al 2008), so probably does STAU1 binding to the CH domain of UPF1. As a second indication that SMD and NMD are in competition, UPF1 that immunoprecipitates with STAU1 is not detectably associated with UPF2, and UPF1 that immunoprecipitates with UPF2 is not detectably associated with STAU1 (Fig.…”

“…In contrast, SMD usually involves translation termination at a normal termination codon and provides a means to conditionally downregulate the expression of genes encoding mRNAs that contain an SBS in their 39 untranslated region (UTR). Notably, NMD, unlike SMD, also involves UPF3X (also called UPF3b) and UPF2 in a process whereby UPF3X anchors UPF2 to EJCs (Lykke-Andersen et al 2000;Serin et al 2001;Singh et al 2007;Chamieh et al 2008).…”

“…Each pathway requires UPF1, which is a member of the RNA helicase superfamily 1 that manifests RNA-dependent ATP hydrolytic and 59-39 ATP-dependent unwinding activities in vitro (Bhattacharya et al 2000;Cheng et al 2007;Chamieh et al 2008). During classical NMD, when translation terminates sufficiently upstream of a post-splicing EJC, UPF1 binding to the EJC via UPF2 is thought to trigger mRNA decay (Lykke-Andersen et al 2000;Kim et al 2005;Kashima et al 2006;Wittmann et al 2006;Singh et al 2007;.…”

Structural insights into nonsense-mediated mRNA decay (NMD) by electron microscopy

“…A molecular link between SURF components and a downstream EJC is required to trigger UPF1 phosphorylation by the SMG1 kinase (indicated as an arrow). UPF2 and UPF3 required to bridge UPF1 and the EJC [17]. SMG1 kinase is regulated by SMG8 and SMG9 [30], but interactions of SMG1 and UPF2 have also been described [36].…”

“…Despite intensive research, we do not fully understanding the molecular basis of this 50-55 nucleotide rule. According to current models, a large complex bridging the ribosome and an EJC, and containing the three UP-Frameshift proteins, UPF1, UPF2 and UPF3, assembles only when a downstream EJC comes into proximity of a ribosome that has terminated prematurely ( Figure 1a) [1,[3][4][5][6][7]17]. UPF1 is an RNA helicase that associates with ribosomes by interacting with eukaryotic release factors (eRFs) eRF1 and eRF3.…”