Characterization of mature mitochondrial transcripts in Drosophila, and the implications for the tRNA punctuation...

The mitochondrial genome of Drosophila melanogaster is thought to be transcribed in about five polycistronic primary transcripts, which are processed into 11 mRNA, 22 tRNA and two rRNA species required for the genome's function. The tRNA punctuation model has been proposed to predict the cleavage sites used in this process. In this model, tRNAs are removed from the primary transcripts and the fragments that remain become mRNA and rRNA transcripts. Thus the 5' and 3' ends of the major gene transcripts are defined by the endpoints of the intervening tRNA sequences. We used 5' and 3' RACE, and circularization and RT-PCR methods to determine the sequences of both ends of all major gene transcripts of the D. melanogaster mitochondrial genome. In general, the tRNA punctuation model accurately predicts the 3' ends of most mRNA and rRNA molecules, even where there are non-coding residues present. Non-coding residues at the 5' end are evidently removed during RNA processing. The mRNAs begin precisely at the start codon for each gene. In particular, the 5' end of the cox1 gene is the first in-frame sense codon, UCG, implying that this codon serves as the start. In-frame TAA stop codons immediately preceding both the cox1 and cox2 genes may serve to prevent in-frame translation of these genes prior to the completion of processing, and are removed from the mature transcripts. Where multiple tRNA genes are present, as between nad3 and nad5, they are removed sequentially in a 3' to 5' direction.