SMG1:SMG8:SMG9-complex integrity supports efficient execution of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. NMD activation depends on phosphorylation of the RNA helicase UPF1 by the SMG1 kinase, which acts in a complex with SMG8 and SMG9. Structural and biochemical studies have implicated SMG8 and SMG9 as regulators of SMG1 activity, but their contributions to NMD in human cells remain incompletely defined. Here, we systematically dissect the roles of SMG8 and SMG9 in NMD using genetic and pharmacological perturbations in multiple human cell lines. Deletion of the kinase inhibitory domain (KID) of SMG8 did not affect UPF1 phosphorylation or NMD efficiency, demonstrating that this domain is dispensable in vivo. Complete loss of SMG8 or SMG9 resulted in only modest NMD impairment and was accompanied by moderately increased UPF1 phosphorylation. However, SMG8- or SMG9-deficient cells exhibited pronounced hypersensitivity to partial pharmacological inhibition of SMG1, leading to synergistic, transcriptome-wide stabilization of NMD targets. These effects were reproducible across different cellular contexts, underscoring a general regulatory role for SMG8 and SMG9. Together, our results establish SMG8 and SMG9 as nonessential modulators that safeguard the efficiency and perturbation tolerance of the NMD pathway in human cells.