N- and C-terminal Upf1 phosphorylations create binding platforms for SMG-6 and SMG-5:SMG-7 during NMD

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors

Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains

The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form. Here, we uncovered that the C-terminal domain of Upf1, conserved in higher eukaryotes and containing several essential phosphorylation sites, also inhibits the flanking helicase domain. With different biochemical approaches we show that this domain, named SQ, directly interacts with the helicase domain to impede ATP hydrolysis and RNA unwinding. The phosphorylation sites in the distal half of the SQ domain are not directly involved in this inhibition. Therefore, in the absence of multiple binding partners, Upf1 is securely maintained in an inactive state by two intramolecular inhibition mechanisms. This study underlines the tight and intricate regulation pathways required to activate multifunctional RNA helicases like Upf1

Structural and functional analysis of the three MIF4G domains of nonsense-mediated decay factor UPF2

Nonsense-mediated decay (NMD) is a eukaryotic quality control pathway, involving conserved proteins UPF1, UPF2 and UPF3b, which detects and degrades mRNAs with premature stop codons. Human UPF2 comprises three tandem MIF4G domains and a C-terminal UPF1 binding region. MIF4G-3 binds UPF3b, but the specific functions of MIF4G-1 and MIF4G-2 are unknown. Crystal structures show that both MIF4G-1 and MIF4G-2 contain N-terminal capping helices essential for stabilization of the 10-helix MIF4G core and that MIF4G-2 interacts with MIF4G-3, forming a rigid assembly. The UPF2/UPF3b/SMG1 complex is thought to activate the kinase SMG1 to phosphorylate UPF1 in vivo. We identify MIF4G-3 as the binding site and in vitro substrate of SMG1 kinase and show that a ternary UPF2 MIF4G-3/UPF3b/SMG1 complex can form in vitro. Whereas in vivo complementation assays show that MIF4G-1 and MIF4G-2 are essential for NMD, tethering assays reveal that UPF2 truncated to only MIF4G-3 and the UPF1-binding region can still partially accomplish NMD. Thus UPF2 MIF4G-1 and MIF4G-2 appear to have a crucial scaffolding role, while MIF4G-3 is the key module required for triggering NMD

Target Discrimination in Nonsense-Mediated mRNA Decay Requires Upf1 ATPase Activity

RNA quality control pathways get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the ATPase cycle of the SF1 Helicase Upf1 is required for mRNA discrimination during Nonsense-Mediated Decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD -translation and termination codon-proximal poly(A) binding protein - depend on the ATPase activity of Upf1 to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase-dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be a widely employed activity in target RNA discrimination