Insights into the recruitment of the NMD machinery from the crystal structure of a core EJC-UPF3b complex

Abstract

In mammals, Up-frameshift proteins (UPFs) form a surveillance complex that interacts with the exon junction complex (EJC) to elicit nonsense-mediated mRNA decay (NMD). UPF3b is the component of the surveillance complex that bridges the interaction with the EJC. Here, we report the 3.4 A resolution crystal structure of a minimal UPF3b-EJC assembly, consisting of the interacting domains of five proteins (UPF3b, MAGO, Y14, eIF4AIII, and Barentsz) together with RNA and adenylyl-imidodiphosphate. Human UPF3b binds with the C-terminal domain stretched over a composite surface formed by eIF4AIII, MAGO, and Y14. Residues that affect NMD when mutated are found at the core interacting surfaces, whereas differences between UPF3b and UPF3a map at peripheral interacting residues. Comparison with the binding mode of the protein PYM underscores how a common molecular surface of MAGO and Y14 recognizes different proteins acting at different times in the same pathway. The binding mode to eIF4AIII identifies a surface hot spot that is used by different DEAD-box proteins to recruit their regulators.

Fig. 1.

Minimal interaction domains in the EJC-UPF3b structure. (A) Schematic representation of the domain arrangements of the human proteins used in this study. Color-filled areas identify structural domains, which include the two RecA-like domains of the DEAD-box protein eIF4AIII (in dark gray), the MAGO protein fold (in blue), the RRM domains of Y14 and UPF3b (in pink and green, respectively), and the three MIF4G domains (middle domain of eukaryotic initiation factor 4G) of UPF2 (in brown). The arrows and corresponding residue numbers indicate the constructs used for the biochemical reconstitution. The portions of the polypeptides ordered in the three-dimensional structure are shaded in light gray.

Fig. 2.

Structure of the EJC-UPF3b complex. (A) View of the structure of the EJC core (eIF4AIII in gray, BTZ in red, MAGO in blue, Y14 in pink, and RNA, AMPPNP, and magnesium in black) in complex with the EJC-binding domain of UPF3b (in green, shown together with the electron density map contoured at 0.9σ). UPF3b residues 418–432 bind in an extended conformation to a composite surface of the EJC comprising MAGO, the RRM of Y14, and the eIF4AIII C-terminal RecA-like domain (domain 2). This and all other ribbon drawings were generated using PYMOL (http://www.pymol.org). The structure is viewed in two orientations related by a counterclockwise rotation of 120° around a vertical axis. (B) Close-up view of the interactions between UPF3b (green) and MAGO-Y14 (blue and pink, respectively), showing the central position of Arg423_UPF3b in the structure. The molecules are viewed in a similar orientation to that used in the left panel of A. (C) Close-up view of the interactions between UPF3b (green) and eIF4AIII (gray), showing the prominent interaction of Tyr429_UPF3b. The molecules are viewed in a similar orientation to that used in the left panel of A.

Fig. 3.

UPF3b conservation and affinity of the interactions. (A) Sequence alignment of the EJC-binding domain of UPF3b. The alignment includes the sequences of the two paralogues (UPF3a and UPF3b) present in Homo sapiens (H.s., Q9H1J1-1 and Q9BZI7-2, respectively) and Danio rerio (D.r., Q7SXF0-1 and B05733-1, respectively), of UPF3b from Mus musculus (M.m., Q3ULJ3) and Drosophila melanogaster (D.m., Q9W1H3-1). Conserved residues are highlighted in green. Above the sequences, colored circles identify residues of human UPF3b that interact with eIF4AIII (gray circles), with MAGO (blue circles) and Y14 (pink circles). Residues of human UPF3b shown by mutagenesis studies to affect NMD are indicated with a star (the filled star indicates the most important residue identified in ref. . (B) A conserved positively charged stretch of UPF3b is involved in NMD. A β-globin reporter with 4boxB in the 3′UTR was cotransfected in HeLa cells with a transfection control and plasmids expressing λN alone, wild-type λN-UPF3b, or different mutant versions of λN-UPF3b as indicated. Northern-blot of total RNAs probed with a specific body-radiolabeled DNA probes for β-globin is shown. β-globin 4boxB mRNA levels were normalized to that of the control (wt + 300 + e3, ref. 13). The normalized value of β-globin 4boxB mRNA level is set to 100% in cells transfected with λN vector. Mean values ± SD are indicated. (C) Binding of the EJC to UPF3b or UPF3a peptides (residues 414–441 and 427–454) in solution as measured by fluorescence anisotropy. The peptides were labeled with fluorescein at the carboxy terminus, and for labeling purposes a serine was introduced in place of the nonconserved cysteine residue at position 440 and 453 in UPF3b and UPF3a (Fig. 3A). The data were fitted to a binding equation describing a single-site binding model. The best fit was plotted as a solid line (green for UPF3b and blue for UPF3a).

Fig. 4.

Protein recognition at similar molecular surfaces. (A) The surfaces of eIF4AIII (gray) and yeast eIF4A (light brown) are shown (after optimal superimposition) bound to the corresponding interacting proteins, UPF3b (green) and eIF4G (dark blue). The close-up view shows the same structural position for Tyr429_UPF3b and Trp579_eIF4G. (B) The surface of MAGO-Y14 is used for binding both UPF3b (green) and PYM (orange). The dark and light shades of MAGO and Y14 correspond to the UPF3b-bound structure and to the PYM-bound structure, respectively. The molecules are shown after optimal superimposition in an orientation that is turned 70° around a vertical axis and 50° degree around a horizontal axis as compared to the orientation in A.