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.
Lohan
Amanda J
AJ
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
Gray
Michael W
MW
D000048:Acanthamoeba / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000235:genetics*; D017393:RNA Editing / Q000235:genetics*; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013395:Sucrose / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D013395:Sucrose; D012313:RNA; D012343:RNA, Transfer