Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation

Abstract

UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.

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

“…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 ]. 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 ].…”

“…UPF3B is a nucleocytoplasmic shuttling protein which binds to the EJC in the nucleus and is then exported to the cytoplasm in complex with the EJC [ 5 ]. Human UPF3B comprises an RNA recognition motif-like (RRM-L) domain in its N-terminal, followed by a NONA/paraspeckle-like (NOPS-L) region, two predicted coiled-coil-like (CCL-1 and CCL-2) regions and a C-terminal EJC-binding motif (EBM) ( Figure 4 ) [ 114 ].…”

“…UPF3B interacts with UPF2′s MIF4G-3 through its RRM-L domain and NOPS-L region [ 114 ]. The crystal structure of the complex revealed an intimate interaction where UPF2′s MIF4G-3 domain wedges between the RRM-L and NOPS-L domains of UPF3B.…”

“…The crystal structure of the complex revealed an intimate interaction where UPF2′s MIF4G-3 domain wedges between the RRM-L and NOPS-L domains of UPF3B. The NOPS-L-mediated interaction with UPF2′s MIF4G-3 is essential for high-affinity binding [ 112 , 114 ]. In fact, the UPF3B Y160D mutation causing neurodevelopmental disease is located in the UPF3B NOPS-L region.…”

“…In fact, the UPF3B Y160D mutation causing neurodevelopmental disease is located in the UPF3B NOPS-L region. On the molecular level, aspartate residue 160 is displaced from a hydrophobic cleft formed by UPF2′s MIF4G-3, causing a ~40-fold decrease in UPF2-UPF3B interaction affinity [ 114 ]. This weakened affinity between UPF3B and UPF2 was shown to lead to an upregulation of UPF3A (see below) [ 117 ] and decreased NMD efficiency, as evidenced by increased mRNA levels of factors regulating neurodevelopment, such as ATF4 and ARHGAP24 [ 65 ].…”