ABSTRACT
tRNAHis guanylyltransferase (Thg1) catalyzes the 3'-5' incorporation of guanosine into position -1 (G-1) of tRNAHis. G-1 is unique to tRNAHis and is crucial for recognition by histidyl-tRNA synthetase (HisRS). Yeast Thg1 requires ATP for G-1 addition to tRNAHis opposite A73, whereas archaeal Thg1 requires either ATP or GTP for G-1 addition to tRNAHis opposite C73. Paradoxically, human Thg1 (HsThg1) can add G-1 to tRNAsHis with A73 (cytoplasmic) and C73 (mitochondrial). As N73 is immediately followed by a CCA end (positions 74-76), how HsThg1 prevents successive 3'-5' incorporation of G-1/G-2/G-3 into mitochondrial tRNAHis (tRNAmHis) through a template-dependent mechanism remains a puzzle. We showed herein that mature native human tRNAmHis indeed contains only G-1. ATP was absolutely required for G-1 addition to tRNAmHis by HsThg1. Although HsThg1 could incorporate more than one GTP into tRNAmHisin vitro, a single-GTP incorporation prevailed when the relative GTP level was low. Surprisingly, HsThg1 possessed a tRNA-inducible GTPase activity, which could be inhibited by ATP. Similar activity was found in other high-eukaryotic dual-functional Thg1 enzymes, but not in yeast Thg1. This study suggests that HsThg1 may downregulate the level of GTP through its GTPase activity to prevent multiple-GTP incorporation into tRNAmHis.
Subject(s)
Nucleotidyltransferases/metabolism , RNA, Transfer, His , Adenosine Triphosphate , GTP Phosphohydrolases/genetics , Guanosine , Guanosine Triphosphate/metabolism , Histidine-tRNA Ligase , Humans , RNA, Transfer , RNA, Transfer, His/genetics , RNA, Transfer, His/metabolism , Saccharomyces cerevisiae/metabolismABSTRACT
The extra 5' guanine nucleotide (G-1) on tRNAHis is a nearly universal feature that specifies tRNAHis identity. The G-1 residue is either genome encoded or post-transcriptionally added by tRNAHis guanylyltransferase (Thg1). Despite Caenorhabditis elegans being a Thg1-independent organism, its cytoplasmic tRNAHis (CetRNAnHis) retains a genome-encoded G-1. Our study showed that this eukaryote possesses a histidyl-tRNA synthetase (CeHisRS) gene encoding two distinct HisRS isoforms that differ only at their N-termini. Most interestingly, its mitochondrial tRNAHis (CetRNAmHis) lacks G-1, a scenario never observed in any organelle. This tRNA, while lacking the canonical identity element, can still be efficiently aminoacylated in vivo. Even so, addition of G-1 to CetRNAmHis strongly enhanced its aminoacylation efficiency in vitro. Overexpression of CeHisRS successfully bypassed the requirement for yeast THG1 in the presence of CetRNAnHis without G-1. Mutagenesis assays showed that the anticodon takes a primary role in CetRNAHis identity recognition, being comparable to the universal identity element. Consequently, simultaneous introduction of both G-1 and the anticodon of tRNAHis effectively converted a non-cognate tRNA to a tRNAHis-like substrate. Our study suggests that a new balance between identity elements of tRNAHis relieves HisRS from the absolute requirement for G-1.
Subject(s)
Caenorhabditis elegans/genetics , Nucleotides/genetics , RNA, Mitochondrial/genetics , RNA, Transfer, His/metabolism , Amino Acid Sequence , Aminoacylation , Animals , Anticodon/genetics , Base Sequence , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Enzyme Stability , Histidine-tRNA Ligase/chemistry , Histidine-tRNA Ligase/genetics , Kinetics , Nucleotidyltransferases , Protein Domains , Protein Isoforms/genetics , Protein Isoforms/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins , Substrate Specificity , TemperatureABSTRACT
BACKGROUND: Brain-derived neurotrophic factor (BDNF) protein or mRNA levels may be involved in the pathophysiology of bipolar disorder. However, the results were inconsistent. We aimed to simultaneously investigate the relationship of BDNF protein and mRNA levels in peripheral blood of patients with bipolar mania. METHODS: Patients with bipolar mania (n = 30) and healthy controls (n = 30) were recruited during our one-year study. Psychiatric diagnoses were made according to Diagnostic and Statistical Manual of Mental Disorders, 4th Edition criteria. The scores of the Young Mania Rating Scale (YMRS) of patients with bipolar mania were greater than 26. All participants had peripheral blood drawn to analyze the serum BDNF protein and mRNA levels. RESULTS: Using t-test, patients with bipolar mania had a lower BDNF protein and mRNA levels than did the healthy controls (p < 0.001 and 0.049, respectively), however, the statistical significances were lost after analysis of co-variance adjusted for age and body mass index. Twenty seven out of 30 patients with bipolar mania remained in the study after the 4 weeks of mood stabilizer treatment. Patients' BDNF protein and mRNA levels did not change significantly after 4-week treatment. CONCLUSIONS: Our study found that serum BDNF protein and mRNA levels in patients with bipolar mania were lower than healthy controls, but a larger sample size will be needed to confirm this finding.
