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 encodes 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 of the mRNA. This results in a predicted protein whose carboxyl 576 amino acids are identical to those of the cytoplasmic enzyme but with a different amino terminus of 49 amino acids that contains a putative mitochondrial targeting sequence. Expression of the two lysyl-tRNA synthetase-green fluorescent protein gene fusions in a human cell line confirmed that the cytoplasmic form was targeted to the cytoplasm and the mitochondrial form to mitochondria. The genomic lysyl-tRNA synthetase gene consisted of 15 exons. The two isoforms were created by alternative splicing of the first three exons of the 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 mitochondrial isoform with an additional upstream small open reading frame, consisting mainly of a portion of the 5' coding region of the cytoplasmic isoform. This is the first example of mitochondrial targeting sequence being encoded on the second exon of a gene. Ribonuclease protection analysis showed that the mRNA encoding the cytoplasmic isoform makes up approximately 70%, and the mitochondrial isoform approximately 30%, of the mature transcripts from the lysyl-tRNA synthetase gene. The mitochondrial form of the enzyme, purified after expression in Escherichia coli, aminoacylated in vitro transcripts corresponding to both the cytoplasmic and mitochondrial tRNA(Lys), despite the difference in the discriminator base sequence in the acceptor stems of these tRNAs.
Tolkunova
E
E
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Park
H
H
Xia
J
J
King
M P
MP
Davidson
E
E
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D003593:Cytoplasm / Q000201:enzymology*; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005091:Exons; D020224:Expressed Sequence Tags; D049452:Green Fluorescent Proteins; D006801:Humans; D007438:Introns; D008164:Luminescent Proteins / Q000378:metabolism; D008250:Lysine-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D018076:DNA, Complementary; D008164:Luminescent Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008250:Lysine-tRNA Ligase