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<p>Nonsense-mediated mRNA decay (NMD) relies on the coordinated assembly and action of multiple protein factors. Degradation of target mRNAs begins with endonucleolytic cleavage near premature stop codons, but the mechanisms of endonuclease activation and regulation remain unclear. Using structural predictions, biochemical in vitro assays, and cell-based NMD analysis, we show that SMG5 and SMG6 interact via their PIN domains to form a composite interface (cPIN) with full endonuclease activity. In vitro reconstituted SMG5-SMG6 cPIN heterodimers show high activity, as SMG5 completes the SMG6 active site and substrate binding site. Mutations in residues at their predicted interaction surfaces, RNA-binding sites, or active&#xa0;site attenuate or abolish cPIN activity in vitro and impair cellular NMD. Our findings demonstrate how paralogous PIN domains complement each other to assemble a highly active endonuclease in NMD, providing a structural and mechanistic explanation for efficient NMD substrate degradation.</p>

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Composite SMG5-SMG6 PIN domain formation is essential for NMD

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年2月19日

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  • Katharina Kurscheidt,
  • Sophie Theunissen,
  • Natalia Pasquali,
  • Kerstin Becker,
  • Volker Boehm,
  • Elena Conti,
  • Niels H. Gehring

摘要

Nonsense-mediated mRNA decay (NMD) relies on the coordinated assembly and action of multiple protein factors. Degradation of target mRNAs begins with endonucleolytic cleavage near premature stop codons, but the mechanisms of endonuclease activation and regulation remain unclear. Using structural predictions, biochemical in vitro assays, and cell-based NMD analysis, we show that SMG5 and SMG6 interact via their PIN domains to form a composite interface (cPIN) with full endonuclease activity. In vitro reconstituted SMG5-SMG6 cPIN heterodimers show high activity, as SMG5 completes the SMG6 active site and substrate binding site. Mutations in residues at their predicted interaction surfaces, RNA-binding sites, or active site attenuate or abolish cPIN activity in vitro and impair cellular NMD. Our findings demonstrate how paralogous PIN domains complement each other to assemble a highly active endonuclease in NMD, providing a structural and mechanistic explanation for efficient NMD substrate degradation.