A Single Editing Event Is a Prerequisite for Efficient Processing of Potato Mitochondrial Phenylalanine tRNA

Abstract

In bean, potato, and Oenothera plants, the C encoded at position 4 (C₄) in the mitochondrial tRNA^Phe_GAA gene is converted into a U in the mature tRNA. This nucleotide change corrects a mismatched C₄-A₆₉ base pair which appears when the gene sequence is folded into the cloverleaf structure. C-to-U conversions constitute the most common editing events occurring in plant mitochondrial mRNAs. While most of these conversions introduce changes in the amino acids specified by the mRNA and appear to be essential for the synthesis of functional proteins in plant mitochondria, the putative role of mitochondrial tRNA editing has not yet been defined. Since the edited form of the tRNA has the correct secondary and tertiary structures compared with the nonedited form, the two main processes which might be affected by a nucleotide conversion are aminoacylation and maturation. To test these possibilities, we determined the aminoacylation properties of unedited and edited potato mitochondrial tRNA^Phe in vitro transcripts, as well as the processing efficiency of in vitro-synthesized potato mitochondrial tRNA^Phe precursors. Reverse transcription-PCR amplification of natural precursors followed by cDNA sequencing was also used to investigate the influence of editing on processing. Our results show that C-to-U conversion at position 4 in the potato mitochondrial tRNA^Phe_GAA is not required for aminoacylation with phenylalanine but is likely to be essential for efficient processing of this tRNA.