Defects in t6A tRNA modification due to GON7 and YRDC mutations lead to Galloway-Mowat syndrome

Arrondel C, Missoury S, Snoek R, Patat J, Menara G, Collinet B, Liger D, Durand D, Gribouval O, Boyer O, Buscara L, Martin G, Machuca E, Nevo F, Lescop E, Braun D, Boschat A, Sanquer S, Guerrera I, Revy P, Parisot M, Masson C, Boddaert N, Charbit M, Decramer S, Novo R, Macher M, Ranchin B, Bacchetta J, Laurent A, Collardeau-Frachon S, van Eerde A, Hildebrandt F, Magen D, Antignac C, van Tilbeurgh H, Mollet G, Nature Communications 10(1) (2019) DOI

SASDFL8 – GON7, the fifth subunit of human KEOPS, bound to LAGE3-OSGEP (EKC/KEOPS complex subunit:Probable tRNA N6-adenosine threonylcarbamoyltransferase)

EKC/KEOPS complex subunit GON7
EKC/KEOPS complex subunit LAGE3
Probable tRNA N6-adenosine threonylcarbamoyltransferase
MWI(0) 59 kDa
MWexpected 63 kDa
VPorod 91 nm3
log I(s) 1.62×10-2 1.62×10-3 1.62×10-4 1.62×10-5
EKC/KEOPS complex subunit GON7 EKC/KEOPS complex subunit LAGE3 Probable tRNA N6-adenosine threonylcarbamoyltransferase small angle scattering data  s, nm-1
ln I(s)
EKC/KEOPS complex subunit GON7 EKC/KEOPS complex subunit LAGE3 Probable tRNA N6-adenosine threonylcarbamoyltransferase Guinier plot ln 1.63×10-2 Rg: 3.1 nm 0 (3.1 nm)-2 s2
(sRg)2I(s)/I(0)
EKC/KEOPS complex subunit GON7 EKC/KEOPS complex subunit LAGE3 Probable tRNA N6-adenosine threonylcarbamoyltransferase Kratky plot 1.104 0 3 sRg
p(r)
EKC/KEOPS complex subunit GON7 EKC/KEOPS complex subunit LAGE3 Probable tRNA N6-adenosine threonylcarbamoyltransferase pair distance distribution function Rg: 3.1 nm 0 Dmax: 11.5 nm

Data validation


Fits and models


log I(s)
 s, nm-1
EKC/KEOPS complex subunit GON7 EKC/KEOPS complex subunit LAGE3 Probable tRNA N6-adenosine threonylcarbamoyltransferase OTHER model

Synchrotron SAXS data from solutions of GON7 bound to LAGE3-OSGEP in 20 mM MES, 200 mM NaCl, 5 mM β-mercaptoethanol, pH 6.5 were collected on the SWING beam line at SOLEIL (Saint-Aubin, France) using a AVIEX PCCD170170 detector at a sample-detector distance of 1.8 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 65.00 μl sample at 1.5 mg/ml was injected at a 0.30 ml/min flow rate onto a Agilent Bio SEC-3, 300 Å column at 15°C. 255 successive 1 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

The scattered intensities were displayed on an absolute scale (cm-1) using the scattering of water. SAXS data were normalized to the intensity of the incident beam, averaged and background subtracted using the program US-SOMO. Frames were examined individually and 21 identical frames were averaged and further processed. The corresponding SEC-elution concentration was approximately 0.35 mg/mL. The complex is constituted of one monomer of OSGEP, one monomer of LAGE3 and one monomer of GON7. Molecular mass was obtained using the Bayesian inference approach proposed in PRIMUS/qt, which combines four concentration independent MM estimators: M=59.5 kDa within the credibility interval [57.5-64.5].

EKC/KEOPS complex subunit GON7 (GON7)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   11.7 kDa
 
UniProt   Q9BXV9 (1-100)
Sequence   FASTA
 
EKC/KEOPS complex subunit LAGE3 (LAGE3)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   14.8 kDa
 
UniProt   1-143 (Q14657)
Sequence   FASTA
 
Probable tRNA N6-adenosine threonylcarbamoyltransferase (OSGEP)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   36.4 kDa
 
UniProt   Q9NPF4 (1-335)
Sequence   FASTA
 
PDB code   6GWJ