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

Abstract

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.

Caspases shutdown nonsense-mediated mRNA decay during apoptosis

“…With respect to UPF2, specific functions have not been correlated with the different regions of UPF2 other than the interaction domain in the C-terminal region with UPF1 and SMG1. 24 This C-terminal region partially retains the UPF2 function in NMD and is consistent with findings showing a dominant negative effect of the N-terminal caspasecleavage UPF2 fragment on NMD, which is not observed with the C-terminal caspase-cleavage fragment ( Figure 8). Our study shows that the N-terminal part of UPF2 has the dual property to induce apoptosis and to inhibit NMD making this region of interest to understand the function of the fulllength UPF2.…”

“…Little is known about the role of UPF2 in NMD; however, a recent study has shown that the first and/or the second MIF4G domains of UPF2 are essential for NMD. 24 Given that the UPF2 caspase-cleavage site is predicted to be located in the second MIF4G making UPF2 nonfunctional during apoptosis (Figure 2). …”

Mechanism, factors, and physiological role of nonsense-mediated mRNA decay

“…The domain architecture of UPF2 consists of three tandem MIF4G domains (middle portion of eIF4G), followed by the U1BD [130][131][132]. Proteins containing MIF4G domains are commonly involved in general mRNA metabolism, such as the components of the nuclear or cytoplasmic cap-binding complex (CBC) CBP80 and eIF4G, or the spliceosomal protein CWC22 [130,[133][134][135][136].…”

“…This interaction establishes a linear cascade ranging from UPF1 over UPF2 to UPF3 [122]. Whereas the exact function of the first two MIF4G domains is not known, a structural role was proposed in which these domains are required to form a ring-like scaffolding structure required for NMD factor assembly [131,139]. Moreover, conserved residues on the surface of the N-terminal helices of MIF4G-1 of the S. cerevisiae Upf2 were shown to be essential for NMD [140].…”

The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay

“…UPF2 binds directly to the C-terminal part of SMG1 and stimulates the phosphorylation of UPF1 (Kashima et al 2006;Clerici et al 2013). Phosphorylated UPF1 recruits the homologous proteins SMG5/SMG7 and SMG6, leading to the degradation of the target mRNA (Okada-Katsuhata et al 2012).…”