SMG1, a PI3K-related kinase, plays a critical role in nonsense-mediated mRNA decay (NMD) in mammals. SMG1-mediated phosphorylation of the UPF1 helicase is an essential step during NMD initiation. Both SMG1 and UPF1 are presumably activated by UPF2, but this regulation is incompletely understood. Here we reveal that SMG1C (a complex containing SMG1, SMG8, and SMG9) contributes to regulate NMD by recruiting UPF1 and UPF2 to distinct sites in the vicinity of the kinase domain. UPF2 binds SMG1 in an UPF1-independent manner in vivo, and the SMG1C-UPF2 structure shows UPF2 recognizes the FRB domain, a region that regulates the related mTOR kinase. The molecular architectures of several SMG1C-UPFs complexes, obtained by combining electron microscopy with in vivo and in vitro interaction analyses, competition experiments, and mutations, suggest that UPF2 can be transferred to UPF1 within SMG1C, inducing UPF2-dependent conformational changes required to activate UPF1 within an SMG1C-UPF1-UPF2 complex.

Running title: Structure of the human UPF1-UPF2-UPF3-EJC complexMelero et al 2 Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF surveillance complex near a PTC. In humans, UPF assembly is prompted by the exon junction complex (EJC). We investigated the molecular architecture of the UPF complex bound to the EJC by cryo-electron microscopy (cryo-EM) and using positional restraints from additional EM, mass spectrometry and biochemical interaction data. The heptameric assembly is built around UPF2, a scaffold protein with a ring structure that closes around the CH domain of UPF1, keeping the helicase region in an accessible and unwinding-competent state. UPF2 also positions UPF3 to interact with the EJC. The geometry is such that this transient complex poises UPF1 to elicit helicase activity towards the 3′ end of the mRNP.The expression of eukaryotic genes is regulated at multiple levels to control the production of functional proteins at the appropriate amount, location and time in different cell types. The modulation of mRNA levels by targeted degradation has emerged as a widespread mechanism to down-regulate gene expression post-transcriptionally. Several pathways mediate the depletion of the translatable pool of physiological and non-physiological transcripts (reviewed in 1,2 ). Nonsense-mediated mRNA decay (NMD) was originally discovered as the surveillance pathway that detects and degrades mRNAs with premature translation termination codons (PTCs) (reviewed in 3,4 ). These aberrant mRNAs arise frequently as a result of germline mutations in inherited genetic disorders, of pre-mRNA processing errors and of nonproductive rearrangements at the DNA or RNA level (reviewed in 5,6 ). NMD also modulates the steady-state level of physiological mRNAs, amounting to about 10 % of the transcriptome (reviewed in 7 ). Melero et al 3The NMD pathway is evolutionary conserved in eukaryotes and essential in humans (reviewed in 8 ). Work over the years in different model organisms has shown that NMD requires translating ribosomes and a combination of cis-acting elements and trans-acting factors to signal whether the context of translation termination is physiological or aberrant 9,10 .Cis-acting elements can originate from the 3′ untranslated region (UTR), whose length and features influence the process of translation termination (the 'faux 3′ UTR' model) 9,11,12 . In addition, a major determinant that promotes NMD in human cells derives from splice junctions 3,4 . Here, four proteins assemble onto mRNA upon splicing to form the exonjunction complex (EJC), a stable constituent of the spliced mRNP 13 . In humans, NMD is elicited when a stop codon is present at least 50-54 nucleotides upstream of a splice junction.This observation, made decades ago, is now interpreted in molecular terms as the requirement of a minimal distance for a ribosome stalled at a PTC to establish the appropriate n...

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.