The PIN domain of SMG-5 functionally interacts with SMG-6 to stimulate NMD

Nonsense-mediated mRNA decay (NMD) is a translational-dependent mRNA decay pathway that regulates mRNAs and protects cells from the deleterious, truncated protein products of mRNAs with early stop codons. Despite substantial effort, the central biochemical reactions that comprise mRNA decay during NMD remain elusive. Research by our laboratory and others centers around the observation that NMD target mRNA cleavage by the endonuclease SMG-6 requires the presence of another NMD factor, SMG-5, although the molecular basis of SMG-6's requirement for SMG-5 remained elusive. Here we present work to explain the requirement of SMG-5 in SMG-6-mediated mRNA cleavage. We revisit previous observations that SMG-5 contains a catalytically inactive PIN nuclease domain, and we show that although SMG-5 lacks conventional active site residues, the PIN domain of SMG-5 nevertheless contains highly conserved residues that are essential to NMD. We show that AlphaFold predicts an interaction between SMG-5 and SMG-6 PIN domains, an interaction that we substantiate via in vitro pulldowns. We use the in silico models to design point mutations that perturb—and restore—NMD function in Caenorhabditis elegans via a compensatory salt bridge flip. Altogether, our data support the idea that SMG-5 and SMG-6 interact to form a functional complex, and we suggest molecular roles for the overlooked SMG-5 PIN domain in SMG-6-mediated mRNA cleavage.