Human histidyl-tRNA synthetase: recognition of amino acid signature regions in class 2a aminoacyl-tRNA synthetases

Abstract

We have determined the sequence of cDNA for the human histidyl-tRNA synthetase (HRS) in a hepatoma cell line and confirmed it in fetal myoblast and fibroblast cell lines. The newly determined sequence differs in 48 places, including insertions and deletions, from a previously published sequence. By sequence specific probing and by direct sequencing, we have established that only the newly determined sequence is present in genomic DNA and we have sequenced 500 hundred bases upstream of the translation start site. The predicted amino acid sequence now clearly demonstrates all three motifs recognized in class 2 aminoacyl-tRNA synthetases. Alignment of E. coli, yeast, and when available, mammalian predicted amino acid sequences for three of the four members of the class 2a subgroup (his, pro, ser, and thr) shows strong preservation of amino acid specific signature regions proximal to motif 2 and proximal to motif 3. These probably represent the active site binding regions for the proximal acceptor stem and for the amino acid. The first two exons of human HRS contain a 32 amino acid helical motif, first described in human QRS, a class 1 synthetase, which is found also in a yeast RNA polymerase, a rabbit termination factor, and both bovine and human WRS, suggesting that it may be an RNA binding motif.

Anti-Ro52 antibodies frequently co-occur with anti-Jo-1 antibodies in sera from patients with idiopathic inflammatory myopathy

“…A cDNA encoding the Jo-1 protein was isolated from a human cDNA library by hybridization with an oligonucleotide derived from the published sequence [28]. DNA sequence analysis showed that this cDNA was almost identical to the cDNA published by Raben et al [28].…”

Critical Requirement for Professional APCs in Eliciting T Cell Responses to Novel Fragments of Histidyl-tRNA Synthetase (Jo-1) in Jo-1 Antibody-Positive Polymyositis

“…Jo-1-specific primers linked to PstI restriction sites were then used for cDNA amplification by PCR; resulting products were digested with PstI and subcloned into the maltose-binding protein (MBP) expression vector pMALc2 (New England Biolabs, Beverly, MA) previously digested with PstI and treated with calf intestinal phosphatase to prevent religation. Potential clones were subjected to automated sequencing by the University of Pittsburgh core sequencing facility, and resulting products were compared with the previously published sequence for human Jo-1 (26).…”

Genomic organization, transcriptional mapping, and evolutionary implications of the human bi-directional histidyl-tRNA synthetase locus (HARS/HARSL)

“…Nested amplifications of HARSL and HARS transcripts using primers positioned downstream of the corresponding translational start codons (+13-36 and +125-148 relative to the (+1)ATG, respectively) detected multiple mRNA species with varying lengths of 5 0 -UTR mapping within the shared bi-directional promoter region (Fig. 2, lanes 2 Previous reports have suggested that in addition to the short class of mRNAs mapping immediately upstream of the HARS ORF, a second class of longer HARS transcripts may derive from a more distal, independent promoter overlapping the first exon of the HARSL gene [8,16]. We performed an RLM-RACE analysis using primers mapping downstream of this putative transcript and successfully amplified a single mRNA species mapping 352 bp upstream of the HARS ORF (Fig.…”

“…Unlike other ''housekeeping'' genes, the organization and expression of the human HisRS gene is intimately associated with a close structural homologue, HARSL, which is transcribed in the opposite direction from a shared bi-directional promoter. While the function of the HARSL polypeptide is unknown, it is clear that its genomic organization has been preserved since the estimated divergence of humans and rodents approximately [8,16], a similar amplification was performed using nested HARS gene-specific primers mapping approximately 275 bp upstream of the HARS ORF (lane 1). Nested amplification products were size-fractionated in 2% TBE agarose gels, stained with ethidium bromide, and subsequently detected with a fluorescent scanner.…”

“…In addition to the multiple transcripts mapping immediately upstream of the HARS ORF, a second class of longer HARS mRNAs (approximately 380 and 455 bp 5 0 -UTRs) presumably initiating from more distal promoter elements has been reported [8,16]. Using RLM-RACE, we selectively amplified a HARS transcript initiating 352 bp upstream of the HARS ORF and overlapping the opposite strand of HARSL exon 1.…”