Active site assembly by SMG5 as a mechanism for SMG6 endonuclease licensing in nonsense-mediated mRNA decay

Abstract Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that eliminates transcripts containing premature termination codons (PTCs). While the recognition phase of NMD is well characterised, the effector step – how targeted mRNAs are nucleolytically degraded – remains incompletely understood. In metazoans, NMD employs an endonucleolytic route mediated by SMG6, a PIN-domain nuclease, alongside SMG5 and SMG7, which act downstream of PTC recognition. SMG5 has recently been proposed to license SMG6 activity, yet the molecular basis of this licensing has remained elusive. Here, we combine AlphaFold structural predictions with biochemical assays to investigate interactions among human SMG5, SMG6, and SMG7. Structural models predict a high-confidence interface between SMG5 and SMG6 PIN domains that forms a composite active site: a conserved SMG5 aspartate (D893) complements the SMG6 acidic triad to reinstate the canonical tetrad required for PIN-domain catalysis. In vitro, SMG6 alone exhibits weak endonucleolytic activity, which is enhanced ∼10-fold by SMG5 PIN. Mutational analyses confirm that conserved residues from both proteins are essential for this composite configuration. Our findings reveal that the SMG5 PIN domain, previously considered catalytically inert, plays a critical role in activating SMG6 by completing its active site. This work provides mechanistic insight into the SMG5-dependent licensing step and uncovers a composite PIN nuclease architecture at the heart of the metazoan NMD effector phase. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵* shared first authorship e-mail addresses: enessalih.arpa{at}unil.ch, michael.taschner{at}unil.ch, mara.dematos{at}unil.ch Figure 1A, 6E and S4 have been added and some textual edits have been made.