SMG6 is the catalytic endonuclease that cleaves mRNAs containing nonsense codons in metazoan

Abstract

Messenger RNAs harboring nonsense codons (or premature translation termination codons [PTCs]) are degraded by a conserved quality-control mechanism known as nonsense-mediated mRNA decay (NMD), which prevents the accumulation of truncated and potentially harmful proteins. In Drosophila melanogaster, degradation of PTC-containing messages is initiated by endonucleolytic cleavage in the vicinity of the nonsense codon. The endonuclease responsible for this cleavage has not been identified. Here, we show that SMG6 is the long sought NMD endonuclease. First, cells expressing an SMG6 protein mutated at catalytic residues fail to degrade PTC-containing messages. Moreover, the SMG6-PIN domain can be replaced with the active PIN domain of an unrelated protein, indicating that its sole function is to provide endonuclease activity for NMD. Unexpectedly, we found that the catalytic activity of SMG6 contributes to the degradation of PTC-containing mRNAs in human cells. Thus, SMG6 is a conserved endonuclease that degrades mRNAs terminating translation prematurely in metazoa.

SMG6 interacts with the exon junction complex via two conserved EJC-binding motifs (EBMs) required for nonsense-mediated mRNA decay

“…SMG6 mutants lacking the PIN domain or carrying mutations in catalytic residues were also inactive in complementation assays, as shown before (Fig. 1B,D , lanes 13,14;Huntzinger et al 2008). The effect of deleting the SMG6 linker region could not be tested because this mutant was expressed at low levels and was extensively degraded (data not shown).…”

“…The N terminus, upstream of the 14-3-3-like domain, is predicted to be unstructured and is less conserved among SMG6 orthologs. To investigate which domains of SMG6 are important for NMD activity, we designed a series of deletion mutants lacking each domain individually and tested them in a complementation assay (described in Huntzinger et al 2008). Briefly, an siRNA complementary to the ORF of the smg6 mRNA was used to deplete endogenous SMG6; this depletion inhibits NMD.…”

“…It is now well established in D. melanogaster and human cells that SMG6 mediates degradation of NMD targets (Gatfield and Izaurralde 2004;Huntzinger et al 2008;Eberle et al 2009). Previous work showed SMG6 contains a C-terminal PIN domain that provides endonucleolytic activity and a 14-3-3-like domain that likely binds phosphorylated UPF1 and eventually promotes its dephosphorylation (Denning et al 2001;Pal et al 2001;Yamashita et al 2001;Anders et al 2003;Chiu et al 2003;Ohnishi et al 2003;Fukuhara et al 2005;Wittmann et al 2006).…”

“…Plasmids expressing wild-type SMG6, SMG6-PIN-Mut, SMG6DPIN, and MBP were described before (Huntzinger et al 2008). A plasmid …”

“…SMG6 is characterized by a C-terminal PIN (PilT N terminus) domain, which exhibits nuclease activity (Glavan et al 2006). Recent studies in Drosophila melanogaster, zebrafish, and human cells indicate that degradation of NMD targets requires the endonucleolytic activity of SMG6 (Gatfield and Izaurralde 2004;Huntzinger et al 2008;Eberle et al 2009;Wittkopp et al 2009). Nevertheless, a number of key questions remain: For example, how is SMG6 recruited to NMD targets?…”