Analysis of 5′- or 3′-Terminal tRNA Editing: Mitochondrial 5′ tRNA Editing in Acanthamoeba castellanii as the Exemplar

Editing processes that result in the structural retailoring of the aminoacyl acceptor stems of mitochondrial tRNAs are the focus of this chapter. This type of tRNA editing is the most frequently observed and widely distributed and involves nucleotide replacement within the 5′ or 3′ half of the aminoacyl acceptor stem in either a template-directed or a template-independent fashion. We provide a detailed protocol that allows demarcation of 5′-terminal tRNA editing events from those occurring on the 3′ side of the acceptor stem. We present the mitochondrial 5′ tRNA editing system in Acanthamoeba castellanii as the exemplar of terminal tRNA editing. The methodology involves RNA ligase-mediated circularization of tRNAs, cDNA synthesis primed by tRNA-specific oligonucleotides, amplification of cDNA via polymerase chain reaction, and cloning and sequencing of multiple products. This approach permits (1) simultaneous determination of 5′ and 3′ acceptor stem sequences, (2) delineation of 5′ or 3′ editing, (3) identification of mature tRNAs (characterized by having a 3′-CCA_OH motif), (4) identification of processing/editing intermediates, and (5) mechanistic insights.