Yeast mutants accumulating pre-tRNAs [32] initially provided access to substrates suitable for studying tRNA processing in biochemical experiments [33, 34] . These studies led to the conclusion that splicing of pre-tRNAs occurs in two steps. During an endonuclease reaction, the intron is first excised and the resulting tRNA exon halves are then ligated to form a mature tRNA ( Fig. 2A) [35]. The biochemical fractionation of extracts catalyzing pre-tRNA cleavage has led to the identification of tRNA endonuclease proteins in archaea [36] and yeast [37] , facilitating the characterization of the homologous human proteins [38] .