The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript

Two cDNAs encoding human lysyl-tRNA synthetase have been identified. One en codes the cytoplasmic form of the enzyme identified previously. The second cDNA contains the same sequence but with a 180-bp insertion at the 5'-end o f the mRNA. This results in a predicted protein whose carboxyl 576 amino ac ids are identical to those of the cytoplasmic enzyme but with a different a mino terminus of 49 amino acids that contains a putative mitochondrial targ eting sequence. Expression of the two lysyl-tRNA synthetase-green fluoresce nt protein gene fusions in a human cell line confirmed that the cytoplasmic form was targeted to the cytoplasm and the mitochondrial form to mitochond ria, The genomic lysyl-tRNA synthetase gene consisted of 15 exons. The two isoforms were created by alternative splicing of the first three exons of t he gene. The cytoplasmic form was created by splicing exon 1 to exon 3, The inclusion of exon 2 between exons 1 and 3 produced an mRNA encoding the mi tochondrial isoform with an additional upstream small open reading frame, c onsisting mainly of a portion of the 5' coding region of the cytoplasmic is oform, This is the first example of mitochondrial targeting sequence being encoded on the second exon of a gene. Ribonuclease protection analysis show ed that the mRNA encoding the cytoplasmic isoform makes up approximately 70 %, and the mitochondrial isoform approximately 30%, of the mature transcrip ts from the lysyl-tRNA synthetase gene. The mitochondrial form of the enzym e, purified after expression in Escherichia coil, aminoacylated in vitro tr anscripts corresponding to both the cytoplasmic and mitochondrial tRNA(Lys) , despite the difference in the discriminator base sequence in the acceptor stems of these tRNAs.