The ubiquitin-related modifier Urm1 can be covalently conjugated to lysine residues of other proteins, such as yeast Ahp1 and human MOCS3, through a mechanism involving the E1-like protein Uba4 (MOCS3 in humans). Similar to ubiquitination, urmylation requires a thioester intermediate and forms isopeptide bonds between Urm1 and its substrates. In addition, the urmylation process can be significantly enhanced by oxidative stress. Recent findings have demonstrated that Urm1 also acts as a sulfur carrier in the thiolation of eukaryotic tRNA via a mechanism that requires the formation of a thiocarboxylated Urm1. This role is very similar to that of prokaryotic sulfur carriers such as MoaD and ThiS. Evidence strongly supports the hypothesis that Urm1 is the molecular fossil in the evolutionary link between prokaryotic sulfur carriers and eukaryotic ubiquitin-like proteins. In the present review, we discuss the dual role of Urm1 in protein and tRNA modification.
Animals ; Humans ; Models, Biological ; RNA, Transfer ; metabolism ; Sulfur ; metabolism ; Ubiquitin ; metabolism ; Ubiquitins ; metabolism

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

In this study, we aimed to construct a non-replication mRNA platform and explore the side effects of electroporation-mediated delivery of mRNA on the mice as well as the expression features of the mRNA. With luciferase gene as a marker, in vitro transcription with T7 RNA polymerase was carried out for the synthesis of luciferase-expressed mRNA, followed by enzymatic capping and tailing. The mRNA was delivered in vivo by electroporation via an in vivo gene delivery system, and the expression intensity and duration of luciferase in mice were observed via an in vivo imaging system. The results demonstrated that the mRNA transcripts were successfully expressed both in vitro and in vivo. The electroporation-mediated delivery of mRNA had no obvious side effects on the mice. Luciferase was expressed successfully in all the mRNA-transduced mice, while the expression intensity and duration varied among individuals. Overall, the expression level peaked on the first day after electroporation and rapidly declined on the fourth day. This study is of great importance for the construction of non-replication mRNAs and their application in vaccine or antitumor drug development.
Animals ; Electroporation/methods* ; Gene Transfer Techniques ; Luciferases/metabolism* ; Mice ; RNA, Messenger/genetics*

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.
Acetic Acid ; DNA-Binding Proteins/metabolism* ; Fermentation ; Leucine ; RNA, Transfer/genetics* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism* ; Transcription Factors

tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases. However, their exact presence and function in hepatocellular carcinoma (HCC) remain unclear. Here, differentially expressed tsRNAs in HCC were profiled. A novel tsRNA, tRNA^Gln-TTG derived 5'-tiRNA-Gln, is significantly downregulated, and its expression level is correlated with progression in patients. In HCC cells, 5'-tiRNA-Gln overexpression impaired the proliferation, migration, and invasion in vitro and in vivo, while 5'-tiRNA-Gln knockdown yielded opposite results. 5'-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. The suppressed downregulated proteins include ARAF, MEK1/2 and STAT3, causing the impaired signaling pathway related to HCC progression. Furthermore, based on the construction of a mutant 5'-tiRNA-Gln, the sequence of forming intramolecular G-quadruplex structure is crucial for 5'-tiRNA-Gln to strongly bind EIF4A1 and repress translation. Clinically, 5'-tiRNA-Gln expression level is negatively correlated with ARAF, MEK1/2, and STAT3 in HCC tissues. Collectively, these findings reveal that 5'-tiRJNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular G-quadruplex structure, and this process partially inhibits translation and HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Eukaryotic Initiation Factor-4A/genetics* ; Cell Line ; RNA, Transfer/metabolism* ; RNA ; Cell Proliferation

High-throughput RNA-seq has revolutionized the process of small RNA (sRNA) discovery, leading to a rapid expansion of sRNA categories. In addition to the previously well-characterized sRNAs such as microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNA (snoRNAs), recent emerging studies have spotlighted on tRNA-derived sRNAs (tsRNAs) and rRNA-derived sRNAs (rsRNAs) as new categories of sRNAs that bear versatile functions. Since existing software and pipelines for sRNA annotation are mostly focused on analyzing miRNAs or piRNAs, here we developed the sRNA annotation pipelineoptimized for rRNA- and tRNA-derived sRNAs (SPORTS1.0). SPORTS1.0 is optimized for analyzing tsRNAs and rsRNAs from sRNA-seq data, in addition to its capacity to annotate canonical sRNAs such as miRNAs and piRNAs. Moreover, SPORTS1.0 can predict potential RNA modification sites based on nucleotide mismatches within sRNAs. SPORTS1.0 is precompiled to annotate sRNAs for a wide range of 68 species across bacteria, yeast, plant, and animal kingdoms, while additional species for analyses could be readily expanded upon end users' input. For demonstration, by analyzing sRNA datasets using SPORTS1.0, we reveal that distinct signatures are present in tsRNAs and rsRNAs from different mouse cell types. We also find that compared to other sRNA species, tsRNAs bear the highest mismatch rate, which is consistent with their highly modified nature. SPORTS1.0 is an open-source software and can be publically accessed at https://github.com/junchaoshi/sports1.0.
Animals ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing ; Mice ; MicroRNAs ; chemistry ; metabolism ; Molecular Sequence Annotation ; RNA, Ribosomal ; chemistry ; metabolism ; RNA, Small Interfering ; chemistry ; metabolism ; RNA, Small Untranslated ; chemistry ; metabolism ; RNA, Transfer ; chemistry ; metabolism ; Sequence Analysis, RNA ; methods ; Software

Short interfering RNA (siRNA) is widely used for studying post-transcriptional gene silencing and holds great promise as a tool for both identifying function of novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which were designed against different specific areas of coding region of the same target green fluorescent protein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice (Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Fraser fir [Abies fraseri (Pursh) Poir; AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in the bombarded transgenic cells between two siRNAs, and these results were consistent with the inactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis in tested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be the siRNA specific in different plant species. These results indicate that siRNA is a highly specific tool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could be a reliable approach for large-scale screening of gene function and drug target validation.
Cells, Cultured ; Gene Transfer Techniques ; Green Fluorescent Proteins ; genetics ; metabolism ; Plants ; genetics ; metabolism ; RNA Interference ; physiology ; RNA, Small Interfering ; metabolism ; Time Factors

Methods for analyses of protein-protein interactions include: yeast two hybrid (Y2H), phage dis- play (PD), co-immunoprecipitation (Co-IP), glutathione S-transferase pull-down (GST pull-down), cellular co-localization, far-western blotting, virus overlay protein binding assay (VOPBA), surface plasmon resonance (SPR), and fluorescence resonance energy transfer (FRET). Technologies for the detection of protein-nucleic acid interactions include: yeast one hybrid (Y1H), chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), Southwestern blotting, reporter gene, Co-IP, GST pull-down, and PD. These methods are often used in the study of the human enterovirus A71 (EV-A71) by our research team. Reviews in the Chinese literature in this field are lacking, so we reviewed applications of these methods in the study of EV-A71. This review may impart important knowledge in the research of other viruses with regard to protein-protein and protein-nucleic acid interactions.
Electrophoretic Mobility Shift Assay ; Enterovirus A, Human ; chemistry ; genetics ; metabolism ; Fluorescence Resonance Energy Transfer ; RNA, Viral ; metabolism ; Two-Hybrid System Techniques ; Viral Proteins ; metabolism

RNA interference (RNAi) is a powerful endogenous process initiated by short double stranded RNAs, which results in sequence-specific posttranscriptional gene silencing. Because any protein that causes or contributes to a disease is susceptible to RNAi, the RNAi has high potential for therapeutic treatments. In a clinical setting, however, there are many obstacles to targeted delivery of small interfering RNA (siRNA) in vivo, specificity and stability of the RNAi reagents. In this review, we focus on recent progress in the development of efficient siRNA delivery vehicles to help the application of siRNA to in vivo therapy.
Drug Carriers ; Gene Transfer Techniques ; Humans ; Molecular Targeted Therapy ; methods ; trends ; RNA Interference ; RNA, Small Interfering ; administration & dosage ; genetics ; metabolism ; Transfection

The aberrant epigenetic reprogramme is an important cause for abnormal development of nuclear transfer embryos. The objective of this study was to investigate the CpG island methylation profiles and relative expression levels of H19 gene in different tissues of cloned goat fetus. We detected liver, placenta, kidney, lung and heart in the dead cloned goat fetus and the age-matched normal goat fetus (control) by using bisulfite sequencing and real time PCR. Results indicated that methylation levels of the fifth CpG island of H19 gene in dead cloned goat fetus was significant high compared with that in the control in placenta (70% vs 49.41%, P < 0.05), and relative expression levels of H19 gene was significant low compared with that in the control (883.3 vs 1 264.5, P < 0.05). Reversely, the methylation levels was significant low compared with that in the control in lung (63.53% vs 88.24%, P < 0.05), and relative expression levels was significant high compared with that in the control (1 003.4 vs 515.5, P < 0.05). The differences of others groups were insignificant (P > 0.05). Results showed the abnormal DNA methylation proflies of H19 gene occurred in some tissues of cloned goat fetus, which affected normal expression levels of H19 gene, indicating that aberrant DNA methylation reprogramme may be one of the important factors for the death of cloned animals.
Animals ; Cloning, Organism ; veterinary ; CpG Islands ; DNA Methylation ; Epigenesis, Genetic ; Female ; Fetus ; metabolism ; Genomic Imprinting ; Goats ; Kidney ; embryology ; metabolism ; Liver ; embryology ; metabolism ; Lung ; embryology ; metabolism ; Nuclear Transfer Techniques ; RNA, Long Noncoding ; RNA, Untranslated ; genetics ; metabolism