Isolation of two cDNAs encoding functional human cytoplasmic cysteinyl-tRNA synthetase.
Cysteinyl-tRNA synthetase catalyzes the addition of cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. To gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyl-tRNA synthetase. In this study, we sought to clone and express the full-length gene for the human cytoplasmic cysteinyl-tRNA synthetase. Although a gene encoding the human enzyme has been described, the predicted protein sequence, consisting of 638 amino acids, lacks homology with other eukaryotic enzymes in the carboxyl-terminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a full-length cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyl-tRNA synthetases in the carboxyl-terminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyl-tRNA synthetase contained an insertion of 8 bases in the 3' coding region of the mRNA. This insertion arises from an alternative splicing between the last two exons of the gene. The alternative splicing alters the reading frame and results in the replacement of the carboxy-terminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the full-length and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA(Cys) with cysteine.
Davidson
E
E
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Caffarella
J
J
Vitseva
O
O
Hou
Y M
YM
King
M P
MP
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007527:Isoenzymes; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase