Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5–SMG7 and SMG6

Abstract

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes mRNAs with premature stop codons and targets them for rapid degradation. Evidence from previous studies has converged on UPF1 as the central NMD factor. In human cells, the SMG1 kinase phosphorylates UPF1 at the N-terminal and C-terminal tails, in turn allowing the recruitment of the NMD factors SMG5, SMG6 and SMG7. To understand the molecular mechanisms, we recapitulated these steps of NMD in vitro using purified components. We find that a short C-terminal segment of phosphorylated UPF1 containing the last two Ser-Gln motifs is recognized by the heterodimer of SMG5 and SMG7 14–3–3-like proteins. In contrast, the SMG6 14–3–3-like domain is a monomer. The crystal structure indicates that the phosphoserine binding site of the SMG6 14–3–3-like domain is similar to that of SMG5 and can mediate a weak phospho-dependent interaction with UPF1. The dominant SMG6–UPF1 interaction is mediated by a low-complexity region bordering the 14–3–3-like domain of SMG6 and by the helicase domain and C-terminal tail of UPF1. This interaction is phosphorylation independent. Our study demonstrates that SMG5–SMG7 and SMG6 exhibit different and non-overlapping modes of UPF1 recognition, thus pointing at distinguished roles in integrating the complex NMD interaction network.

Mechanism, factors, and physiological role of nonsense-mediated mRNA decay

“…SQ and TQ motifs in the extended and unstructured Nand C-terminus of UPF1 are the preferred motifs for phosphorylation by SMG1 [105,106,154]. Even though phosphorylation was also reported for yeast Upf1, the mechanism and the responsible kinase are different.…”

“…The most studied isoforms of NMD factors were chosen for representation and match, except for SMG1, those indicated in Table 2 (see footnote b for SMG1 discrepancy). UPF1 phosphorylation sites (SQ and TQ motifs) verified by various experimental approaches (ultradeep HeLa cell phosphoproteome [222], in vitro phophorylation assay with UPF1 peptides [106] or full length UPF1 [154] ) are indicated. The phosphosites connected to specific recruiting functions are highlighted specifically Mechanism, factors, and physiological role of nonsense-mediated mRNA decay invertebrates.…”

“…The 14-3-3-like domain of SMG7 is mostly responsible for the phosphorylation-dependent interaction between phosphorylated amino acids (e.g. S1096) in the C-terminus of UPF1 and the heterodimer SMG5-SMG7 [154,157,159,161]. The 14-3-3-like domain of SMG5, which by itself is not able to interact with UPF1, is postulated to provide additional binding strength and specificity [159,161].…”

“…Like SMG5 and SMG7, SMG6 contains a 14-3-3-like domain, which is located centrally in the protein [157]. Interestingly, the SMG6 14-3-3-like domain was found to be monomeric as it neither forms homodimers nor heterodimers with either SMG5 or SMG7 14-3-3-like domains [154]. This domain was also suggested to bind phosphorylated UPF1 and biochemical analysis showed that mutation of the residues in the phosphopeptide binding pocket abolished the interaction with UPF1 [161].…”

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways

“…The endonuclease SMG6 is recruited to NMD-targeted transcripts by activated UPF1 (Okada-Katsuhata et al 2012;Chakrabarti et al 2014;Nicholson et al 2014) and cleaves them in the vicinity of the TC (Huntzinger et al 2008;Eberle et al 2009;Boehm et al 2014;LykkeAndersen et al 2014;Schmidt et al 2015). For the second pathway, the SMG5/SMG7 heterodimer binds to phosphorylated SQ epitopes in the C-terminal part of UPF1 and recruits through the C terminus of SMG7 the deadenylase CCR4/NOT Loh et al 2013;Chakrabarti et al 2014). Whether the SMG6-and the SMG7-mediated decay pathways act independently of each other and maybe even target a distinct subpopulation of mRNAs has so far not been addressed on endogenous targets at a genomewide level.…”