In vitro characterization of a tRNA editing activity in the mitochondria of Spizellomyces punctatus, a chytridiomycete fungus

In the chytridiomycete fungus, Spizellomyces punctatus, all eight of the mitochondrially encoded tRNAs are predicted to have one or more base pair mismatches at the first three positions of their aminoacyl acceptor stems. These tRNAs are edited post-transcriptionally by replacement of the 5'-nucleotide in each mismatched pair with a nucleotide that can form a standard Watson-Crick base pair with its counterpart in the 3'-half of the stem. The type of mitochondrial tRNA editing found in S. punctatus also occurs in Acanthamoeba castellanii, a distantly related amoeboid protist. Using an S. punctatus mitochondrial extract, we have developed an in vitro assay of tRNA editing in which nucleotides are incorporated into various tRNA substrates. Experiments employing synthetic transcripts revealed that the S. punctatus tRNA editing activity incorporates nucleotides on the 5'-side of substrate tRNAs, uses the W-sequence as a template for incorporation, and adds nucleotides in a 3'-to-5' direction. This activity can add nucleotides to a triphosphorylated 5'-end in the absence of ATP but requires ATP to add nucleotides to a monophosphorylated 5'-end; moreover, it functions independently of the state of tRNA 3' processing. These data parallel results obtained in a previous in vitro study of A. castellanii tRNA editing, suggesting that remarkably similar activities function in the mitochondria of these two organisms. The evolutionary origins of these activities are discussed.