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 i nsertions that are absent from bacterial sequences. To gain insights into t he differences between the bacterial and eukaryotic forms, we previously st udied the E. coli cysteinyl-tRNA synthetase. In this study, we sought to cl one and express the full-length gene for the human cytoplasmic cysteinyl-tR NA synthetase. Although a gene encoding the human enzyme has been described , the predicted protein sequence, consisting of 638 amino acids, lacks homo logy with other eukaryotic enzymes in the carboxyl-terminus. This suggested that a further investigation was necessary to obtain the definitive sequen ce 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 synthetase s in the carboxyl-terminus. We also found that approximately 20% of the mRM A encoding the cytoplasmic cysteinyl-tRNA synthetase contained an insertion of 8 bases in the 3 ' coding region of the mRNA, This insertion arises fro m an alternative splicing between the last two exons of the gene. The alter native 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. c oli generated functional proteins that were active in aminoacylation of hum an cytoplasmic tRNA(Cys) with cysteine.