Journal: Nucleic Acids Res / Year: 2015 Title: A network of SMG-8, SMG-9 and SMG-1 C-terminal insertion domain regulates UPF1 substrate recruitment and phosphorylation. Authors: Aurélien Deniaud / Manikandan Karuppasamy / Thomas Bock / Simonas Masiulis / Karine Huard / Frédéric Garzoni / Kathrin Kerschgens / Matthias W Hentze / Andreas E Kulozik / Martin Beck / ...Authors: Aurélien Deniaud / Manikandan Karuppasamy / Thomas Bock / Simonas Masiulis / Karine Huard / Frédéric Garzoni / Kathrin Kerschgens / Matthias W Hentze / Andreas E Kulozik / Martin Beck / Gabriele Neu-Yilik / Christiane Schaffitzel / Abstract: Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD ...Mammalian nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that degrades mRNAs containing premature translation termination codons. Phosphorylation of the essential NMD effector UPF1 by the phosphoinositide-3-kinase-like kinase (PIKK) SMG-1 is a key step in NMD and occurs when SMG-1, its two regulatory factors SMG-8 and SMG-9, and UPF1 form a complex at a terminating ribosome. Electron cryo-microscopy of the SMG-1-8-9-UPF1 complex shows the head and arm architecture characteristic of PIKKs and reveals different states of UPF1 docking. UPF1 is recruited to the SMG-1 kinase domain and C-terminal insertion domain, inducing an opening of the head domain that provides access to the active site. SMG-8 and SMG-9 interact with the SMG-1 C-insertion and promote high-affinity UPF1 binding to SMG-1-8-9, as well as decelerated SMG-1 kinase activity and enhanced stringency of phosphorylation site selection. The presence of UPF2 destabilizes the SMG-1-8-9-UPF1 complex leading to substrate release. Our results suggest an intricate molecular network of SMG-8, SMG-9 and the SMG-1 C-insertion domain that governs UPF1 substrate recruitment and phosphorylation by SMG-1 kinase, an event that is central to trigger mRNA decay.
History
Deposition
Jun 26, 2015
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Header (metadata) release
Jul 8, 2015
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Map release
Jul 15, 2015
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Update
Sep 9, 2015
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Current status
Sep 9, 2015
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_3066.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
contour level in chimera
Voxel size
X=Y=Z: 1.86 Å
Density
Contour Level
By AUTHOR: 0.047 / Movie #1: 0.047
Minimum - Maximum
-0.12514399 - 0.66809916
Average (Standard dev.)
0.0002945 (±0.03465053)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
160
160
160
Spacing
160
160
160
Cell
A=B=C: 297.6 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.86
1.86
1.86
M x/y/z
160
160
160
origin x/y/z
0.000
0.000
0.000
length x/y/z
297.600
297.600
297.600
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-80
0
-4
NX/NY/NZ
161
13
58
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
160
160
160
D min/max/mean
-0.125
0.668
0.000
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Supplemental data
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Sample components
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Entire : Complex of four proteins: SMG1, SMG8, SMG9 and UPF1
Entire
Name: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1
Components
Sample: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1
Protein or peptide: SMG-1
Protein or peptide: SMG-8
Protein or peptide: SMG-9
Protein or peptide: UPF1
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Supramolecule #1000: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1
Supramolecule
Name: Complex of four proteins: SMG1, SMG8, SMG9 and UPF1 / type: sample / ID: 1000 Details: The sample was monodisperse, and elution volume in size exclusion chromatography is consistent with an heterotetramer. Oligomeric state: heterotetramer / Number unique components: 4
pH: 9 Details: 20 mM Tris pH 9.0, 100 mM KCl, 25 mM glycine, 1 mM DTT and 2 mM biotin
Staining
Type: NEGATIVE / Details: cryo
Grid
Details: copper 300 mesh
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: 2 second blotting and blot force of 3
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Electron microscopy
Microscope
FEI POLARA 300
Temperature
Average: 86 K
Specialist optics
Energy filter - Name: not used
Date
Jan 30, 2013
Image recording
Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 1300 / Average electron dose: 13 e/Å2 / Bits/pixel: 32
Tilt angle min
0
Tilt angle max
0
Electron beam
Acceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
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