The tRNA structure contains conserved modifications that are responsible for its stability and are involved in the initiation and accuracy of the translation process. tRNA modification enzymes are prevalent in bacteria, archaea, and eukaryotes. tRNA Gm18 methyltransferase (TrmH) and tRNA m1G37 methyltransferase (TrmD) are prevalent and essential enzymes in bacterial populations. TrmH involves itself in methylation process at the 2'-OH group of ribose at the 18th position of guanosine (G) in tRNAs. TrmD methylates the G residue next to the anticodon in selected tRNA subsets. Initially, m1G37 modification was reported to take place on three conserved tRNA subsets (tRNA(Arg), tRNA(Leu), tRNA(Pro)); later on, few archaea and eukaryotes organisms revealed that other tRNAs also have the m1G37 modification. The present study reveals Gm18, m1G37 modification, and positions of m1G that take place next to the anticodon in tRNA sequences. We selected extremophile organisms and attempted to retrieve the m1G and Gm18 modification bases in tRNA sequences. Results showed that the Gm18 modification G residue occurs in all tRNA subsets except three tRNAs (tRNA(Met), tRNA(Pro), tRNA(Val)). Whereas the m1G37 modification base G is formed only on tRNA(Arg), tRNA(Leu), tRNA(Pro), and tRNA(His), the rest of the tRNAs contain adenine (A) next to the anticodon. Thus, we hypothesize that Gm18 modification and m1G modification occur irrespective of a G residue in tRNAs.
Adenine ; Anticodon ; Archaea ; Bacteria ; Eukaryota ; Guanosine ; Methylation ; Ribose ; RNA, Transfer* ; RNA, Transfer, Arg ; RNA, Transfer, His ; RNA, Transfer, Leu ; RNA, Transfer, Pro

The 2 species of the genus Anoplocephala (Anoplocephalidae), A. perfoliata and A. magna, are among the most important equine cestode parasites. However, there is little information about their differences at the molecular level. The present study revealed that the mitochondrial (mt) genome of A. magna was 13,759 bp in size and 700 bp shorter than that of A. perfoliata. The 2 species includes 2 rRNA, 22 tRNA, and 12 protein-coding genes each. The size of each of the 36 genes was the same as that of A. perfoliata, except for cox1, rrnL, trnC, trnS2(UCN), trnG, trnH, trnQ, and trnP. In the full mitochondrial genome, the sequence similarity was 87.1%. The divergence in the nucleotide and amino acid sequences of individual protein-coding genes ranged from 11.1% to 16% and 6.8% to 16.4%, respectively. The 2 noncoding regions of the mt genome of A. magna were 199 bp and 271 bp in length, while the equivalent regions in A. perfoliata were 875 bp and 276 bp, respectively. The results of this study support the proposal that A. magna and A. perfoliata are separate species, consistent with previous morphological analyses.
Amino Acid Sequence ; Cestoda ; Genome ; Genome, Mitochondrial* ; Parasites ; RNA, Transfer

BACKGROUND: The genus Candida comprises 163 species, the most common pathogen in the genus is C. albicans, however, other Candida species are considered as emergent pathogens. Because there are species-specific differences in the susceptibility of Candida spp. to the currently used therapeutic drugs, species identification is critical for therapeutic planning and accurate epidemiological records. The objective of this study is to evaluate the performance of tRNA intergenic length polymorphism (tDNA-ILP) analysis for the accurate identification of Candida species. METHODS: For the identifying the 7 type strains and 54 clinical isolates of Candida, the suitability of tDNA-ILP was evaluated. The polymerase chain reaction (PCR) using a pair of primers or a single primer was performed. The PCR products were separated by electrophoresis in 2% agarose gels for 70 min. RESULTS: In seven Candida type strains, tDNA-ILP using a single primer (reverse) can be easily analyzed by visual comparision. Fifty-three of 54 strains were identified as the same species with conventional identification. CONCLUSIONS: The tDNA-ILP analysis can be useful for the simple and rapid identification of Candida species in routine laboratories.
Candida* ; Electrophoresis ; Gels ; Polymerase Chain Reaction ; RNA, Transfer ; Sepharose

tRNA-derived RNA fragments (tRFs) are an emerging class of non-coding RNAs (ncRNAs). A growing number of reports have shown that tRFs are not random degradation products but are functional ncRNAs made of specific tRNA cleavage. They play regulatory roles in several biological contexts such as cancer, innate immunity, stress responses, and neurological disorders. In this review, we summarize the biogenesis and functions of tRFs.
Organelle Biogenesis* ; Humans* ; Immunity, Innate ; Nervous System Diseases ; Neurodegenerative Diseases ; RNA* ; RNA, Transfer ; RNA, Untranslated

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
Animals ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Oryzias/genetics* ; RNA, Guide/genetics* ; RNA, Transfer/genetics*

In vitro transcription systems with T7 RNA polymerase (T7 RNAP) were widely used in preparation of RNA because of their simplicity and high efficiency. The transcripts would have additional 5' sequence since T7 promoter spans the transcription start site, while deletion of the transcription start site would severely reduce the T7 RNAP transcriptional activity. We successfully developed an in vitro transcription by combining of T7 RNAP high efficient transcription system and highly specific self-splicing technology of ribozymes, in this system, ribozyme self-splices at the designed specific site and releases the aim RNA without affecting transcription efficiency of T7 RNAP, the aminoacylation activity of human mitochondrial tRNA(Trp) (HmtRNA(Trp) (UCA)) is 113.6 pmol/microg. This method with its high efficiency on transcription and good repeatability is very suitable for preparation of accurate RNA in large scale.
Base Sequence ; DNA-Directed RNA Polymerases ; genetics ; Humans ; Molecular Sequence Data ; RNA ; genetics ; RNA Splicing ; RNA, Catalytic ; genetics ; RNA, Transfer, Trp ; genetics ; Transcription, Genetic ; Transfer RNA Aminoacylation ; genetics ; Viral Proteins ; genetics

OBJECTIVE: To explore the correlation of mitochondrial DNA (mtDNA) variants and coronary heart disease (CHD) in a Chinese pedigree and the possible molecular mechanisms. METHODS: A Chinese pedigree featuring matrilineal inheritance of CHD who visited Hangzhou First People's Hospital in May 2022 was selected as the study subject. Clinical data of the proband and her affected relatives was collected. By sequencing the mtDNA of the proband and her pedigree members, candidate variants were identified through comparison with wild type mitochondrial genes. Conservative analysis among various species was conducted, and bioinformatics software was used to predict the impact of variants on the secondary structure of tRNA. Real-time PCR was carried out to determine the copy number of mtDNA, and a transmitochondrial cell line was established for analyzing the mitochondrial functions, including membrane potential and ATP level. RESULTS: This pedigree had contained thirty-two members from four generations. Among ten maternal members, four had CHD, which yielded a penetrance rate of 40%. Sequence analysis of proband and her matrilineal relatives revealed the presence of a novel m.4420A>T variant and a m.10463T>C variant, both of which were highly conserved among various species. Structurally, the m.4420A>T variant had occurred at position 22 in the D-arm of tRNAMet, which disrupted the 13T-22A base-pairing, while the m.10463T>C variant was located at position 67 in the acceptor arm of tRNAArg, a position critical for steady-state level of the tRNA. Functional analysis revealed that patients with the m.4420A>T and m.10463T>C variants exhibited much fewer copy number of mtDNA and lower mitochondrial membrane potential (MMP) and ATP contents (P < 0.05), which were decreased by approximately 50.47%, 39.6% and 47.4%, respectively. CONCLUSION Mitochondrial tRNAMet 4420A>T and tRNAArg 10463T>C variants may underlay the maternally transmitted CHD in this pedigree, which had shown variation in mtDNA homogeneity, age of onset, clinical phenotype and other differences, suggesting that nuclear genes, environmental factors and mitochondrial genetic background have certain influence on the pathogenesis of CHD.
Humans ; Female ; Mutation ; Pedigree ; RNA, Transfer, Met ; East Asian People ; RNA, Transfer, Arg ; DNA, Mitochondrial/genetics* ; Coronary Disease/genetics* ; Adenosine Triphosphate

OBJECTIVE: Gonadotropin hyperstimulation for in vitro fertilization and embryo transfer (IVF-ET) has been considered to be harmful to the uterine receptivity. The role of HB-EGF has been implicated in the mouse embryonic implantation. In the present study, we assessed the effect of gonadotropin hyperstimulation on the expression of HB-EGF mRNA in murine endometrium. METHODS: Competitive, reverse transcription-PCR was performed to evaluate the expression of HB-EGF mRNA in the hyperstimulated murine endometrum from pregnant uteri of gestation day (g.d.) 5.5 after the injection of saline (placebo), pregnant mare serum gonadotropin (PMSG) 5 IU and 10 IU. RESULTS: HB-EGF expression was observed at 10-4-10-6 dilution concentrations of internal standard, and dual bands (423/360 bp) were observed at 10-5 dilution concentration. The expression of HB-EGF mRNA showed no significant change in the endometrial expression in PMSG groups, compared to the control group, on g.d. 5.5. There was no dose-response relationship between the two PMSG groups. CONCLUSION: Our data reveals that ovarian hyperstimulation using gonadotropin does not influence the expression of HB-EGF in the murine endometrium. Further studies are necessary to elucidate which stage of embryonic implantation and the expression of other substance is affected by the ovarian hyperstimulation.
Animals ; Embryo Transfer ; Endometrium* ; Female ; Fertilization in Vitro ; Gonadotropins* ; Mice ; Pregnancy ; RNA, Messenger ; Uterus

A high proportion of modified nucleotides has been found in mitochondrial tRNA. Such modification can promote accurate folding of tRNA and its stability, while unmodified mitochondrial tRNA may fold into various 2D-structures with impaired functions. Therefore, modification of mitochondrial tRNA is closely related to mitochondrial diseases. Particularly, positions 9, 34, 37, 54 and 55 of the mitochondrial tRNA are critical for such modification. Mutations at these positions are important cause for mitochondrial dysfunction and have been associated with various mitochondrial diseases.
DNA, Mitochondrial ; chemistry ; genetics ; Humans ; Mitochondrial Diseases ; genetics ; Mutation ; Nucleic Acid Conformation ; RNA, Transfer ; chemistry ; genetics

Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
DNA Helicases ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; RNA, Transfer ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism