Poster abstracts

Poster number 37 submitted by Jiqiang Ling

Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with MERRF syndrome

Jiqiang Ling (Department of Microbiology and the Ohio State Biochemistry Program, the Ohio State University), Herve Roy, Mary Anne T. Rubio (Department of Microbiology, the Ohio State University), Daoming Qin, Juan D. Alfonzo, Kurt Fredrick (Department of Microbiology and the Ohio State Biochemistry Program, the Ohio State University), Michael Ibba (Department of Microbiology and the Ohio State Biochemistry Program, the Ohio State University)

Abstract:
Human mitochondrial tRNA (hmt-tRNA) mutations are associated with a variety of diseases including mitochondrial myopathies, diabetes, encephalopathies, and deafness. Since the current understanding of the precise molecular mechanisms of these mutations is limited, there is no efficient method to treat their associated mitochondrial diseases. Here, we utilize a variety of known mutations in hmt-tRNAPhe to investigate the mechanisms that lead to malfunctions. We tested the impact of hmt-tRNAPhe mutations on aminoacylation, structure, and translation elongation factor binding. The majority of the mutants were pleiotropic, exhibiting defects in aminoacylation, global structure and elongation factor binding. One notable exception was the G34A anticodon mutation of hmt-tRNAPhe (mitochondrial DNA mutation G611A), which is associated with myoclonic epilepsy with ragged red fibers (MERRF). In vitro the G34A mutation decreases aminoacylation activity by 100-fold, but does not affect global folding or recognition by elongation factor. Furthermore, G34A hmt-tRNAPhe does not undergo adenosine-to-inosine (A-to-I) editing ruling out mis-coding as a possible mechanism for mitochondrial malfunction. To improve the aminoacylation state of the mutant tRNA, we modified the tRNA binding domain of the nucleus-encoded human mitochondrial phenylalanyl-tRNA synthetase, which aminoacylates hmt-tRNAPhe with cognate phenylalanine. This variant enzyme displayed significantly improved aminoacylation efficiency for the G34A mutant, suggesting a general strategy to treat certain classes of mitochondrial diseases by modification of the corresponding nuclear gene.

References:
Ling,J., Roy,H., Qin,D., Rubio,M.A., Alfonzo,J., Fredrick,K., & Ibba,M. Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with MERRF syndrome. Proc. Natl. Acad. Sci. USA. In press.

Keywords: tRNA, mitochondrial disease, gene therapy