A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

Abstract

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activities.

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

“…However, the unstructured region preceding the 14-3-3-like domain of SMG6 was observed to bind UPF1 in a phospho-independent manner in vitro [154]. This observation was supported by functional studies of SMG6 tethering and UPF1 complementation assays performed in another recent publication [179]. Additionally, two EBMs were characterized in the very N-terminus of SMG6, which, similarly to UPF3b, mediate the interaction with the EJC [145].…”

“…Mutations of any of the catalytic aspartate residues, which are required to coordinate divalent metal ions for the nucleophilic attack of H 2 O on the phosphodiester bond of the RNA, renders the protein inactive and abolishes endonucleolytic degradation of NMD targets [55,145,174,[177][178][179]. Like SMG5 and SMG7, SMG6 contains a 14-3-3-like domain, which is located centrally in the protein [157].…”

Nonsense-mediated mRNA decay in humans at a glance

“…Phosphorylation of UPF1 also enhances its interaction with SMG6 and/or the SMG5−SMG7 heterodimer (Franks et al, 2010;Kashima et al, 2006;Kurosaki et al, 2014;Ohnishi et al, 2003), although a phosphorylation-independent interaction between UPF1 and SMG6 has also been reported (Chakarabarti et al, 2014;Nicholson et al, 2014) (see poster). SMG5 and SMG6, which possess a PilT-Nterminal (PIN) domain that typifies some ribonucleases, bind to phosphorylated UPF1 and promote its dephosphorylation through the recruitment of protein phosphatase 2A, apparently after mRNA decay initiates Boehm et al, 2014;Chiu et al, 2003;Kurosaki et al, 2014;Lee et al, 2015;Ohnishi et al, 2003;Schmidt et al, 2015) (see poster).…”

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

“…In mammalian cells, two mechanistically distinct pathways have been described to execute the degradation of the target mRNAs (Mühlemann and Lykke-Andersen 2010). 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).…”