Endonucleolytic cleavage is the primary mechanism of decay elicited by nonsense-mediated mRNA decay.

Premature stop codon-containing mRNAs can produce truncated and dominantly acting proteins that harm cells. Eukaryotic cells protect themselves by degrading such mRNAs via the nonsense-mediated mRNA decay (NMD) pathway. The precise reactions by which cells attack NMD-target mRNAs remain obscure, precluding a biochemical understanding of NMD and hampering therapeutic efforts to control NMD. Here, we modify and deploy single-molecule nanopore mRNA sequencing to clarify the route by which NMD targets are attacked in an animal. We obtain single-molecule measures of splicing isoform, cleavage state, and poly(A) tail length. We observe robust endonucleolytic cleavage of NMD targets in vivo that depends on the nuclease SMG-6. We show that NMD-target mRNAs experience deadenylation and decapping, similar to that of normal mRNAs. Furthermore, we show that a factor (SMG-5) that historically was ascribed a function in deadenylation and decapping is in fact required for SMG-6-mediated cleavage. Our results support a model in which NMD factors act in concert to degrade NMD targets in animals via an endonucleolytic cleavage near the stop codon, and we suggest that deadenylation and decapping are normal parts of mRNA (and NMD target) maturation and decay rather than unique facets of NMD.