Modulation of RNA-binding properties of the RNA helicase UPF1 by its activator UPF2

Abstract

The NMD helicase UPF1 is a prototype of the superfamily 1 (SF1) of RNA helicases that bind RNA with high affinity and translocate on it in an ATP-dependent manner. Previous studies showed that UPF1 has a low basal catalytic activity that is greatly enhanced upon binding of its interaction partner, UPF2. Activation of UPF1 by UPF2 entails a large conformational change that switches the helicase from an RNA-clamping mode to an RNA-unwinding mode. The ability of UPF1 to bind RNA was expected to be unaffected by this activation mechanism. Here we show, using a combination of biochemical and biophysical methods, that binding of UPF2 to UPF1 drastically reduces the affinity of UPF1 for RNA, leading to a release of the bound RNA. Although UPF2 is capable of binding RNA in vitro, our results suggest that dissociation of the UPF1-RNA complex is not a consequence of direct competition in RNA binding but rather an allosteric effect that is likely mediated by the conformational change in UPF1 that is induced upon binding its activator. We discuss these results in light of transient interactions forged during mRNP assembly, particularly in the UPF1-dependent mRNA decay pathways.

Nonsense-Mediated mRNA Decay Factor Functions in Human Health and Disease

“…In this UPF2-independent ‘branch’ of EJC-dependent NMD ( Figure 1 ), CASC3 is suggested to directly interact with eIF4A3, UPF3B as well as UPF1, with UPF3B binding to UPF1 and eRF3, thereby stabilising UPF1 in the vicinity of the EJC, despite of the lack of UPF2 [ 48 , 49 , 50 ]. In the canonical NMD pathway, UPF2 promotes UPF1’s ATPase and helicase activities and, thereby, supports NMD activation [ 51 , 52 , 53 ]. Recent studies have shed light on the mechanisms compensating for the absence of UPF2 in UPF2-independent NMD, revealing the involvement of serine/threonine-protein kinases AKT ( Figure 1 ) [ 54 , 55 ].…”

“…Structural data of UPF1, in complex with the C-terminus of UPF2, shows that the CH domain of UPF1 is displaced upon UPF2-binding into an open conformation [ 52 , 79 ], resulting in decreased RNA-clamping and increased RNA-unwinding by UPF1 [ 51 , 52 ]. Recent biochemical and biophysical studies have shown that binding of UPF2 to UPF1 directly promotes dissociation of UPF1 from RNA in a non-competitive manner [ 53 ]. A low-resolution cryo-EM structure of the EJC, in complex with RNA and the three UPF proteins, shows that UPF1 is found in the vicinity of the RNA 3′-end [ 93 ].…”

“…In a recent study, UPF1-UPF2 complexes were shown to have a low RNA affinity, despite the fact that both NMD factors have been shown to interact with RNA [ 53 ]. The authors performed fluorescence anisotropy experiments and reported affinities in the low nanomolar range for UPF2 MIF4G domains 1-2, as well as MIF4G domain 3 with the U1BD [ 53 ].…”

“…In a recent study, UPF1-UPF2 complexes were shown to have a low RNA affinity, despite the fact that both NMD factors have been shown to interact with RNA [ 53 ]. The authors performed fluorescence anisotropy experiments and reported affinities in the low nanomolar range for UPF2 MIF4G domains 1-2, as well as MIF4G domain 3 with the U1BD [ 53 ]. This corroborates previous work showing that UPF2′s MIF4G domain 3 binds RNA in electrophoretic mobility shift assays (EMSAs) via conserved basic and aromatic residues [ 112 ] and work showing ribosome and RNA binding of UPF2 in complex with UPF3B [ 113 , 114 ].…”

“…This corroborates previous work showing that UPF2′s MIF4G domain 3 binds RNA in electrophoretic mobility shift assays (EMSAs) via conserved basic and aromatic residues [ 112 ] and work showing ribosome and RNA binding of UPF2 in complex with UPF3B [ 113 , 114 ]. In complex with UPF1, UPF2 was shown to help UPF1-RNA dissociation, likely by binding to a region allosteric of UPF1’s RNA-binding site [ 53 ].…”