Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2 - PubMed (original) (raw)

Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2

Marcello Clerici et al. EMBO J. 2009.

Abstract

Nonsense-mediated decay (NMD) is a eukaryotic quality control mechanism that degrades mRNAs carrying premature stop codons. In mammalian cells, NMD is triggered when UPF2 bound to UPF3 on a downstream exon junction complex interacts with UPF1 bound to a stalled ribosome. We report structural studies on the interaction between the C-terminal region of UPF2 and intact UPF1. Crystal structures, confirmed by EM and SAXS, show that the UPF1 CH-domain is docked onto its helicase domain in a fixed configuration. The C-terminal region of UPF2 is natively unfolded but binds through separated alpha-helical and beta-hairpin elements to the UPF1 CH-domain. The alpha-helical region binds sixfold more weakly than the beta-hairpin, whereas the combined elements bind 80-fold more tightly. Cellular assays show that NMD is severely affected by mutations disrupting the beta-hairpin binding, but not by those only affecting alpha-helix binding. We propose that the bipartite mode of UPF2 binding to UPF1 brings the ribosome and the EJC in close proximity by forming a tight complex after an initial weak encounter with either element.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Structure of the UPF1(115–914)–UPF2(1105–1198) complex. (A) Ribbon diagram of the complete structure, with UPF1 in green and UPF2 in blue. The missing links between the UPF1 CH- and helicase domains and between the N and C-terminal parts of UPF2 are represented as dotted lines. (B) Superposition of the closed form of the helicase domain (orthorhombic crystal, green) with the previously described helicase domain in the phosphate-bound form (PDB ID 2gk7, blue). The RMSD between the two structures is 1.03 Å for 591 aligned Cα atoms. The RMSD values between the orthorhombic form and the AMPPNP (PDB ID 2gjk) and ADP (PDB ID 2gk6) forms are, respectively, 1.81 and 2.00 Å. (C) Superposition of the open form of the helicase domain (monoclinic crystal, red) with the previously described helicase domain in the ADP-bound form (PDB ID 2gk6, gold). The RMSD between the two structures is 1.35 Å for 584 aligned Cα atoms. The RMSD values between the monoclinic form and the phosphate and AMPPNP forms are, respectively, 2.48 and 3.07 Å. (D) Superposition of UPF1 from the monoclinic (red) and orthorhombic (green) crystal forms showing that the relative orientations of the CH and helicase domains are the same in each case, although the helicase conformation is different. (E) The principal interacting residues from the CH- (green) and helicase (yellow) domains of UPF1 are represented as sticks. The same interactions are found in both monoclinic and orthorhombic crystal forms. These residues are well conserved (Supplementary Figure S2).

Figure 2

Figure 2

UPF2 binds on two opposite surfaces of the UPF1 CH domain. (A, B) UPF2 (blue) is represented as ribbons and UPF1 (grey) as ribbons and a transparent surface. UPF1 zinc atoms are shown in green. The UPF2 missing linker is represented as a dotted line. The two views differ by a rotation of 180 degrees around the horizontal axis. (C) The principal residues of the UPF2 N-terminal helix (cyan) and the UPF1 CH-domain (yellow), which form the hydrophobic interface between the two molecules, are represented as sticks. (D) The main interacting residues of UPF2 C-terminal β-hairpin (cyan) and UPF1 CH domain (yellow) are represented as sticks. Met 1169 and Met 1190 do not interact directly with UPF1 binding but form part of a small hydrophobic core important for the stability of the bound form of UPF2. (E) Sequence alignment of the UPF1-binding domain of representative UPF2 proteins from yeast to human. Residues with similarity >70% are displayed in red. The secondary structure of the UPF1-bound human UPF2 is indicated as α (alpha-helix) and β (beta-strand). Red and blue triangles indicate the main UPF1-interacting residues belonging to the N-terminal helical region and the C-terminal β-hairpin, respectively. The alignment was generated with ClustalX (Thompson et al, 2002) and showed using ESPript (

http://espript.ibcp.fr/ESPript/ESPript/

).

Figure 3

Figure 3

Changes in the UPF1 CH domain on UPF2 binding. Superposition of the UPF1 CH domain in the UPF2 complex (green) and alone (blue, PDB code 2iyk). UPF2 is shown in red. The distal region, notably loop L7, rotates to allow UPF2 binding, notably β-strand addition, whereas the helicase proximal end, including the N- and C-terminal parts, is unchanged.

Figure 4

Figure 4

Interaction of UPF2 with the CH-domain of UPF1 studied using NMR. Overlay of 1H, 15N HSQC spectra of the 15N-labelled UPF1 CH domain (residues 115–287), free (green) and in complex with unlabelled UPF2 comprising (A) the complete bipartite binding motif (residues 1105–1207), (B) the helical motif (residues 1105–1129) and (C) the β-hairpin motif (residues 1167–1207) (blue). The corresponding complexes are indicated schematically. Assigned chemical-shift perturbations are mapped onto the surface of UPF1 and are coloured in red. Cyan residues could not be analysed because of a lack of NMR signal.

Figure 5

Figure 5

Mutational analysis of the UPF2–UPF1 interface. (A) In vitro binding of UPF2 mutants. His-tagged UPF2(1105–1207) mutants were co-expressed with UPF1(115–914) and loaded on Ni2+ resin. The resin was washed with 10 CV of buffer containing 50 mM imidazole, 2 CV of buffer containing 100 mM imidazole and the proteins eluted with buffer containing 500 mM imidazole and analysed on a 10–16% SDS–PAGE. Contaminants are indicated with asterisks. The small UPF2 fragments run slightly differently because of charge variations among the mutants. (B) In vivo mutations and NMD tethering assay. Northern blot analysis of RNA from HeLa cells that were transfected with vectors for the 6MS2 reporter (6MS2) and the control (ctrl), together with MS2 (cp, lane 1), MS2-tagged UPF2 (lane 2) or the indicated mutants of UPF2 (lanes 3–8). (C) Cytoplasmic extracts from cells used in (A) were analysed with an MS2-specific antibody to visualize MS2-UPF2, or with a GFP-specific antibody to visualize the co-transfected GFP. (D) Northern blot analysis of RNA from HeLa cells transfected with Luciferase siRNA (negative control, lanes 1–2) or with a UPF2-targeting siRNA (lanes 3–16). The NMD reporter plasmids β-globin wt or NS39 and a transfection efficiency control (Gehring et al, 2003) were transfected, together with a plasmid expressing the indicated siRNA-insensitive mutants of UPF2 (lanes 5–16). The numbers indicate changes in mRNA abundance±s.d. determined by the analysis of five independent experiments. (E) Immunoblot analysis of the UPF2 expression in the lysate from cells used in (D) with a UPF2-specific antibody; actin served as control for comparable loading.

Similar articles

Cited by

References

    1. Amrani N, Ganesan R, Kervestin S, Mangus DA, Ghosh S, Jacobson A (2004) A faux 3′-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay. Nature 432: 112–118 - PubMed
    1. Azzalin CM, Lingner J (2006) The double life of UPF1 in RNA and DNA stability pathways. Cell Cycle 5: 1496–1498 - PubMed
    1. Behm-Ansmant I, Izaurralde E (2006) Quality control of gene expression: a stepwise assembly pathway for the surveillance complex that triggers nonsense-mediated mRNA decay. Genes Dev 20: 391–398 - PubMed
    1. Behm-Ansmant I, Kashima I, Rehwinkel J, Sauliere J, Wittkopp N, Izaurralde E (2007) mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett 581: 2845–2853 - PubMed
    1. Bhattacharya A, Czaplinski K, Trifillis P, He F, Jacobson A, Peltz SW (2000) Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay. RNA 6: 1226–1235 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources