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

Abstract

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.

Genetic studies of alcohol dependence in the context of the addiction cycle

“…CARS encodes cysteinyl-tRNA synthetase, an enzyme that catalyzes the addition of cysteine to its cognate tRNA (Davidson et al, 2001) (Fig. 1 and Table 3).…”

Identification of CARS-ALK Fusion in Primary and Metastatic Lesions of an Inflammatory Myofibroblastic Tumor

“…CARS catalyzes the addition of cysteine to its cognate RNA and is ubiquitously expressed. Two transcript variants of CARS cDNA were isolated, resulting from the alternative splicing in the 3'coding region (Davidson et al, 2001). This alternative splicing occurs downstream of the breakpoint identified in IMT and does not affect the sequence of the CARS-ALK chimeric protein.…”

“…This alternative splicing occurs downstream of the breakpoint identified in IMT and does not affect the sequence of the CARS-ALK chimeric protein. Comparison of molecular weights of the human CARS determined by gel filtration chromatography and SDS-PAGE suggested that the enzyme exists as multimers, most likely dimers (Davidson et al, 2001). Thus, CARS likely contributes to the neoplastic transformation by lending an active promoter and mediating the homooligomerization of the chimeric product, which activates ALK similar to other ALK fusions ).…”

Aminoacylation of an unusual tRNACys from an extreme halophile

“…The kinetic parameters of aminoacylation by the reconstituted CysRS were determined to compare the halophilic enzyme with its bacterial and eukaryotic counterparts, represented by the previously studied E. coli and human enzymes (Christian et al 2000;Davidson et al 2001), respectively (Table 1). Here, the halophilic aminoacylation was performed at 2.8 M KCl, using Cl − as the sole source of the anion.…”

“…NRC-1 CysRS with 0.5-8 µM wild-type tRNA and 5-100 µM mutant tRNAs. The parameters for E. coli CysRS (Christian et al 2000) and human CysRS (Davidson et al 2001) were published previously.…”