European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France.
EMBO J. 2009 Aug 5;28(15):2293-306. doi: 10.1038/emboj.2009.175. Epub 2009 Jun 25.
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.
无意义介导的衰变 (NMD) 是一种真核生物质量控制机制，可降解携带过早终止密码子的 mRNA。在哺乳动物细胞中，当与下游外显子连接复合物结合的 UPF2 与与停滞核糖体结合的 UPF1 相互作用时，就会触发 NMD。我们报告了关于 UPF2 的 C 末端区域与完整的 UPF1 之间相互作用的结构研究。通过 EM 和 SAXS 证实的晶体结构显示，UPF1 CH 结构域以固定构象对接在其解旋酶结构域上。UPF2 的 C 末端区域天然无结构，但通过分离的α-螺旋和β发夹元件结合到 UPF1 CH 结构域。α-螺旋区域的结合强度比β发夹弱六倍，而组合元件的结合强度则强 80 倍。细胞测定表明，破坏β发夹结合的突变严重影响 NMD，但仅破坏α-螺旋结合的突变则没有影响。我们提出，UPF2 结合 UPF1 的二部分模式通过与任一元件的初始弱相互作用后形成紧密复合物，从而使核糖体和 EJC 紧密接近。
