Structure of the human KEOPS/tRNA complex and characterization of pathogenic variants responsible for the Galloway Mowat syndrome.

N6-threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t6A) is a universal modification essential for translational accuracy and efficiency. The t6A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. Structures of the subunits and subcomplexes of human KEOPS are known, but knowledge on the detailed interactions with tRNA is lacking. We present here the first structure of complete hKEOPS and of its complex with a substrate tRNA by cryo-electron microscopy. The CAA tail of tRNA is bound to the TPRKB subunit and the anti-codon loop is positioned at the entrance of the catalytic site of OSGEP subunit. The flexibility of the OSGEP-TP53RK interface allows hKEOPS to fit the surface of the tRNA elbow. We recently identified mutations in all genes encoding for proteins of the t6A pathway in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. We here expressed and characterized the majority of the Galloway Mowat mutants. All mutants could be purified at high yields and seem to be stable in vitro. The t6A activity for most of the mutants is above 40% of the WT. Using CRISPR-Cas9 technology we replaced the genes encoding the t6A pathway proteins in yeast by their human homologues. This yeast construct was perfectly viable and produced WT levels of t6A modified tRNA. Using this tool, we observed that most of the GAMOS mutants were viable in yeast and yielded comparable t6A modified tRNA levels. Our data indicate that healthy human cellular development depends on an optimized level of t6A tRNA modification and is not compatible with a total loss of function of the t6A machinery.