Methods for Analysis of Mitochondrial tRNA Editing in Acanthamoeba castellanii

We present here methodology for assaying 5′-terminal editing of mitochondrial tRNAs in the amoeboid protist Acanthamoeba castellanii . This type of editing involves replacement of one or more nucleotides within the first three positions at the 5′ end of a tRNA substrate. The assay procedure involves RNA ligase-mediated joining of the 5′ and 3′ ends of a tRNA, use of the resulting circularized tRNA as template for cDNA synthesis primed by tRNA-specific primers over a region that encompasses the ligated 5′ and 3′ halves of the acceptor stem, amplification of cDNA via polymerase chain reaction, and cloning and sequencing of double-stranded cDNA product. This approach has the advantage that it simultaneously reveals potential editing events on the 5′ and 3′ side of an acceptor stem, as well as serves to identify mature tRNAs (characterized by having a 3′-CCA_OH motif) and partially processed intermediates.