Expression of mitochondrial protein-coding genes in Tetrahymena pyriformis

In the ciliate protozoon, Tetrahymena pyriformis, mitochondrial protein-coding genes are highly divergent in sequence, and in a number of cases they lack AUG initiation codons. We asked whether RNA editing might be acting to generate protein sequences that are more conventional than those inferred from the corresponding gene sequences, and/or to create standard AUG initiation codons where these are absent. However, comparison of genomic and cDNA sequences (the latter generated by reverse transcriptase sequencing of T. pyriformis mitochondrial mRNAs) yielded no evidence of mitochondrial RNA editing in this organism. To delineate the 5' ends of mitochondrial protein-coding transcripts, primer extension experiments were conducted. In all cases, 5' termini were found to map within a few nucleotides of potential initiation codons, indicating that T. pyriformis mitochondrial mRNAs have little or no 5' untranslated leader sequence. The pattern of strong primer extension stops suggested that both standard (AUG) and non-standard (AUU, AUA, GUG, UUG) initiation codons are utilized by the Tetrahymena mitochondrial translation system. We also investigated expression of the nad1 gene, which in both T. pyriformis and Paramecium aurelia is split into two portions that are encoded by and transcribed from different DNA strands. Northern hybridization analysis showed that the corresponding transcripts are not trans-spliced, implying that separate N-terminal and C-terminal portions of Nad1 are made in this system. Finally, in a search for primary transcripts, we isolated from a T. pyriformis mitochondrial fraction several small RNAs that were reproducibly labeled by incubation in the presence of [alpha-(32)P]GTP and guanylyltransferase. Partial sequence information revealed that none of these cappable RNAs is encoded in the T. pyriformis mitochondrial genome.