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Isolation of Two cDNAs Encoding Functional Human Cytoplasmic Cysteinyl-tRNA Synthetase

Abstract CysteinyltRNA synthetase catalyzes the addition cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyltRNA synthetase. In this study, we sought to clone and express the fulllength gene for the human cytoplasmic cysteinyltRNA 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 carboxylterminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a fulllength cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyltRNA synthetases in the carboxylterminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyltRNA synthetase contained 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 the replacement of the carboxyterminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the fulllength and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA with cysteine.