Journal
SCIENCE CHINA-LIFE SCIENCES
Volume 63, Issue 8, Pages 1227-1239Publisher
SCIENCE PRESS
DOI: 10.1007/s11427-019-1619-x
Keywords
aminoacyl-tRNA synthetase; tRNA; aminoacylation; protein synthesis
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Funding
- National Key Research and Development Program of China [2017YFA0504000]
- National Natural Science Foundation of China [91940302, 31500644, 31570792, 31822015, 81870896, 31670801]
- Strategic Priority Research Program of the Chinese Academy of Sciences [XDB19010203]
- Shanghai Key Laboratory of Embryo Original Diseases [Shelab201904]
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Aminoacyl-tRNA synthetases (aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases (LysRSs). Previously, two mutations (c.1129G>A and c.517T>C) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNA(Lys), with the c.517T>C mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.
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