PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.

The nuclear exosome and its essential co-factor, the RNA helicase MTR4, play crucial roles in several RNA degradation pathways. Besides unwinding RNA substrates for exosome-mediated degradation, MTR4 associates with RNA-binding proteins that function as adaptors in different RNA processing and decay pathways. Here, we identify and characterize the interactions of human MTR4 with a ribosome processing adaptor, NVL, and with ZCCHC8, an adaptor involved in the decay of small nuclear RNAs. We show that the unstructured regions of NVL and ZCCHC8 contain short linear motifs that bind the MTR4 arch domain in a mutually exclusive manner. These short sequences diverged from the arch-interacting motif (AIM) of yeast rRNA processing factors. Our results suggest that nuclear exosome adaptors have evolved canonical and non-canonical AIM sequences to target human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA-binding proteins.

The PI3K-related kinase (PIKK) SMG1 monitors 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 analogue 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.

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