3' UTR length and messenger ribonucleoprotein composition determine endocleavage efficiencies at termination codons.

Nonsense-mediated mRNA decay (NMD) degrades different classes of mRNAs, including transcripts with premature termination codons (PTCs). The NMD factor SMG6 initiates degradation of substrate mRNAs by endonucleolytic cleavage. Here, we aim to delineate the cascade of NMD-activating events that culminate in endocleavage. We report that long 3' UTRs elicit SMG6-mediated endonucleolytic degradation. The presence of an exon-junction complex (EJC) within the 3' UTR strongly stimulates endocleavage in a distance-independent manner. The interaction of SMG6 with EJCs is not required for endocleavage. Whereas the core NMD component UPF2 supports endonucleolytic decay of long 3' UTR mRNAs, it is mostly dispensable during EJC-stimulated endocleavage. Using high-throughput sequencing, we map endocleavage positions of different PTC-containing reporter mRNAs and an endogenous NMD substrate to regions directly at and downstream of the termination codon. These results reveal how messenger ribonucleoprotein (mRNP) parameters differentially influence SMG6-executed endonucleolysis and uncover central characteristics of this phenomenon associated with translation termination.