A post-translational regulatory switch on UPF1 controls targeted mRNA degradation

Abstract

Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptomewide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 39 untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with F€ orster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 39 UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 39 UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.

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). Additionally, SMG6 is an active endonuclease that transiently associates with and cleaves NMD targets in the vicinity of the PTC, approximately where the interaction between phosphorylated UPF1 and the 3′UTR of mRNA occurs (Eberle et al, 2009;Gatfield and Izaurralde, 2004;Huntzinger et al, 2008;Kurosaki et al, 2014;Lykke-Andersen et al, 2014).…”

“…NMD is triggered upon the translation-dependent and highly regulated association of UPF1 with an EJC when translation terminates sufficiently upstream of an EJC so that the terminating ribosome does not physically displace the EJC (see below). UPF2 bound by UPF3 or UPF3X interacts directly with the cysteine-and histidine-rich domain of UPF1, so as to open and activate the UPF1 helicase domain, facilitating mRNA unwinding and protein remodeling, activities that appear to be crucial for mRNA decay (Chamieh et al, 2008;Chakrabarti et al, 2011);Franks et al, 2010;Lykke-Andersen et al, 2000;Mendell et al, 2000;Serin et al, 2001;Kurosaki et al, 2014).…”

“…This promiscuous UPF1 binding is prevented or removed from 5′UTRs and coding regions by scanning and translationally active ribosomes, respectively, but is possible in 3′UTR regions and has been found to correlate with 3′UTR length (Gregersen et al, 2014;Hogg and Goff, 2010;Kurosaki and Maquat, 2013;Lee et al, 2015;Zünd et al, 2013). Steady-state UPF1 undergoes a transient interaction with any available RNA, from which it is released by its ATP-dependent RNA helicase activity (Kurosaki et al, 2014;Lee et al, 2015), and does not detectably form a complex with UPF2 or UPF3X (Kurosaki et al, 2014) (see poster). Importantly, because steady-state UPF1 binds to cellular RNAs promiscuously, and independently of NMD, cellular NMD targets cannot be predicted based on their co-immunoprecipitation with steady-state UPF1 (Gregersen et al, 2014;Zünd et al, 2013).…”

“…Importantly, because steady-state UPF1 binds to cellular RNAs promiscuously, and independently of NMD, cellular NMD targets cannot be predicted based on their co-immunoprecipitation with steady-state UPF1 (Gregersen et al, 2014;Zünd et al, 2013). Instead, cellular NMD targets can be identified based on their binding to phosphorylated UPF1 as discussed below (Kurosaki et al, 2014).…”

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

“…UPF1 binds RNA rather unspecifically and independent of translation (Hogg and Goff 2010;Hurt et al 2013;Zünd et al 2013). Activation of NMD in metazoans involves phosphorylation of UPF1 by SMG1 (Yamashita et al 2001;Kurosaki et al 2014). In mammalian cells, two mechanistically distinct pathways have been described to execute the degradation of the target mRNAs (Mühlemann and Lykke-Andersen 2010).…”