Mutant phenotypes often vary across genetically distinct individuals. To identify the causes of such genetic background effects, we studied differences in gene essentiality across 18 genetically diverse natural yeast strains. We identified 39 genes that were essential in the laboratory reference strain but not in at least one other genetic background, and we mapped and validated the genetic variants that were responsible for the differences in gene essentiality. These variants typically occurred in single modifier genes that tended to differ between genetic backgrounds. The affected genes often indirectly compensated for the loss of the essential gene and identified naturally occurring evolutionary trajectories. Overall, our results highlight the prevalence of changes in gene essentiality in natural populations, as well as the underlying mechanisms. A thorough understanding of the causes of genetic background effects is crucial for the interpretation of genotype-to-phenotype relationships, including those associated with human disease.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that eliminates transcripts containing premature termination codons (PTCs). Substantial progress has been made in defining the transcript features that mark aberrant translation termination for NMD activation, yet key mechanistic steps remain incompletely understood - including how recruitment of the central NMD factor UPF1 is coupled to the downstream effector phase in which targeted mRNAs are nucleolytically degraded. 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 licence SMG6 activity, yet the molecular basis of this licencing 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 the SMG5 PIN domain. 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 licencing step and uncovers a composite PIN nuclease architecture at the heart of the metazoan NMD effector phase.