Poster abstracts

Poster number 46 submitted by Jiqiang Ling

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

Jiqiang Ling (Ohio State Biochemistry Program), Herve Roy (Microbiology department), Daoming Qin (Ohio State Biochemistry Program), Mary Anne T. Rubio (Microbiology department), Juan D. Alfonzo, Kurt Fredrick (Microbiology department, Ohio State Biochemistry Program), Michael Ibba (Microbiology department, 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. (2007) Pathogenic mechanism of a human mitochondrial tRNAPhe mutation associated with myoclonic epilepsy with ragged red fibers syndrome. Proc. Natl. Acad. Sci. USA. 104, 15299-15304.

Keywords: mitochondrial disease, tRNA, aminoacyl-tRNA synthetase