Journal: Structure / Year: 2014 Title: Structures of SMG1-UPFs complexes: SMG1 contributes to regulate UPF2-dependent activation of UPF1 in NMD. Authors: Roberto Melero / Akiko Uchiyama / Raquel Castaño / Naoyuki Kataoka / Hitomi Kurosawa / Shigeo Ohno / Akio Yamashita / Oscar Llorca / Abstract: 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. ...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.
History
Deposition
May 27, 2014
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Header (metadata) release
Jun 18, 2014
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Map release
Jul 9, 2014
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Update
Aug 20, 2014
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Current status
Aug 20, 2014
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Organism: Homo sapiens (human) / Recombinant cell: 293T cells
Sequence
UniProtKB: Serine/threonine-protein kinase SMG1 GO: DNA repair, RNA metabolic process, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay, ATP binding
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Experimental details
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Structure determination
Method
negative staining
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
0.01 mg/mL
Buffer
pH: 7.5 Details: 10 mM HEPES-KOH, 150 mM NaCl, 20% glycerol, 10 mM MgCl2
Staining
Type: NEGATIVE / Details: 1% uranyl formate
Grid
Details: 400 mesh grid with thin carbon support, glow discharged
Vitrification
Cryogen name: NONE / Instrument: OTHER
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Electron microscopy
Microscope
JEOL 1230
Alignment procedure
Legacy - Astigmatism: Objective lens astigmatism was corrected using a TVIPS F416 CMOS and the EM-MENU software (TVIPS)
Date
Oct 23, 2012
Image recording
Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Number real images: 409 / Average electron dose: 15 e/Å2 Details: Using a TVIPS F416 CMOS and the EM-TOOLS software (TVIPS) Bits/pixel: 16
Electron beam
Acceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
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