Abstract
The PI3K-related kinase (PIKK) SMG1 monitors the progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 is impaired by small molecule inhibitors, is reduced by the SMG1 interactors SMG8 and SMG9, and is downregulated by the so-called SMG1 insertion domain. However, the molecular basis for this complex regulatory network has remained elusive. Here, we present cryo-electron microscopy reconstructions of human SMG1-9 and SMG1-8-9 complexes bound to either a SMG1 inhibitor or a non-hydrolyzable ATP analog at overall resolutions ranging from 2.8 to 3.6 Å. These structures reveal the basis with which a small molecule inhibitor preferentially targets SMG1 over other PIKKs. By comparison with our previously reported substrate-bound structure (Langer et al.,2020), we show that the SMG1 insertion domain can exert an autoinhibitory function by directly blocking the substrate-binding path as well as overall access to the SMG1 kinase active site. Together with biochemical analysis, our data indicate that SMG1 autoinhibition is stabilized by the presence of SMG8. Our results explain the specific inhibition of SMG1 by an ATP-competitive small molecule, provide insights into regulation of its kinase activity within the NMD pathway, and expand the understanding of PIKK regulatory mechanisms in general.
Highlights
Nonsense-mediated mRNA decay (NMD) is a co-translational mRNA quality control pathway central to the detection and removal of mRNAs containing premature termination codons as well as to the regulation of many physiological transcripts (Kurosaki, Popp, and Maquat 2019; Karousis and Muhlemann 2019)
Translation termination at a premature stop codon triggers phosphorylation of the RNA helicase UPF1, which enables the recruitment of downstream effectors, in turn resulting in the degradation of the targeted mRNA by ribonucleases
SMG1 is inhibited by a small molecule compound (SMG1i), and in doing so we identified the molecular mechanisms with which SMG1 is down-regulated by its own insertion domain in cis and SMG8 in trans
Summary
Nonsense-mediated mRNA decay (NMD) is a co-translational mRNA quality control pathway central to the detection and removal of mRNAs containing premature termination codons as well as to the regulation of many physiological transcripts (Kurosaki, Popp, and Maquat 2019; Karousis and Muhlemann 2019). Despite the confounding name reflecting a possible evolutionary origin with a lipid kinase (Keith and Schreiber 1995), and in retrospect the ability of some PIKK family members to bind inositol-6-phosphate (Gat et al 2019), all active PIKKs are Ser-Thr protein kinases These large, multidomain enzymes share an overall similar architecture: a C-terminal catalytic module (comprising the so-called FAT, kinase and FATC domains) and an N-. The C-terminal domain of SMG8 remains poorly defined in all available cryo-EM reconstructions (Zhu et al 2019; Gat et al 2019; Langer et al 2020), hindering a molecular understanding of its regulatory role Another portion of the complex reported to downregulate SMG1 kinase activity is the so-called insertion domain, a large 1200-residue region connecting the SMG1 kinase and FATC domains. SMG1 is inhibited by a small molecule compound (SMG1i), and in doing so we identified the molecular mechanisms with which SMG1 is down-regulated by its own insertion domain in cis and SMG8 in trans
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