With the continuous development of messenger RNA (mRNA) technology, mRNA-based drugs have shown broad application prospects in recent years. Since mRNA is easy to be degraded and difficult to enter cells directly, the mRNA delivery vectors have always been one of the focuses in the development of mRNA-based drugs. Although lipid nanoparticles (LNPs) have been widely used for the delivery of mRNA, they tend to accumulate in the liver, and repeated administration can easily induce inflammatory response which leads to tissue damage. Compared with LNPs, virus-like particles (VLPs) have the advantages of high biocompatibility and safety, being expected to offer new solutions for mRNA delivery. Based on the practical application requirements, this review summarized the research progress in VLPs according to the mRNA delivery steps: particle assembly, delivery into cells, and intracellular release. We hope to provide a basis and design ideas for the development of new VLPs as delivery vectors, promote the application of VLPs in mRNA delivery, and provide new possibilities for the research and application of mRNA-based therapeutics.
RNA, Messenger/administration & dosage* ; Humans ; Nanoparticles/chemistry* ; Genetic Vectors ; Lipids/chemistry* ; Drug Delivery Systems/methods* ; Virion ; Animals ; Gene Transfer Techniques ; Liposomes

Although the function of tRNAs in the translational process is well established, it remains controversial whether tRNA abundance is tightly associated with translational efficiency (TE) in mammals. Moreover, how critically the expression of tRNAs contributes to the establishment of tissue-specific proteomes in mammals has not been well addressed. Here, we measured both tRNA expression using demethylase-tRNA sequencing (DM-tRNA-seq) and TE of mRNAs using ribosome-tagging sequencing (RiboTag-seq) in the brain, heart, and testis of mice. Remarkable variation in the expression of tRNA isodecoders was observed among different tissues. When the statistical effect of isodecoder-grouping on reducing variations is considered through permutating the anticodons, we observed an expected reduction in the variation of anticodon expression across all samples, an unexpected smaller variation of anticodon usage bias, and an unexpected larger variation of tRNA isotype expression at amino acid level. Regardless of whether or not they share the same anticodons, the isodecoders encoding the same amino acids are co-expressed across different tissues. Based on the expression of tRNAs and the TE of mRNAs, we find that the tRNA adaptation index (tAI) and TE are significantly correlated in the same tissues but not between tissues; and tRNA expression and the amino acid composition of translating peptides are positively correlated in the same tissues but not between tissues. We therefore hypothesize that the tissue-specific expression of tRNAs might be due to post-transcriptional mechanisms. This study provides a resource for tRNA and translation studies, as well as novel insights into the dynamics of tRNAs and their roles in translational regulation.
Animals ; Mice ; Anticodon/genetics* ; Transcriptome ; Protein Biosynthesis ; RNA, Transfer/chemistry* ; Amino Acids/metabolism* ; Mammals/metabolism*

More than 100 modifications have been found in RNA. Analogous to epigenetic DNA methylation, epitranscriptomic modifications can be written, read, and erased by a complex network of proteins. Apart from N-methyladenosine (mA), N-methyladenosine (mA) has been found as a reversible modification in tRNA and mRNA. mA occurs at positions 9, 14, and 58 of tRNA, with mA58 being critical for tRNA stability. Other than the hundreds of mA sites in mRNA and long non-coding RNA transcripts, transcriptome-wide mapping of mA also identifies >20 mA sites in mitochondrial genes. mA in the coding region of mitochondrial transcripts can inhibit the translation of the corresponding proteins. In this review, we summarize the current understanding of mA in mRNA and tRNA, covering high-throughput sequencing methods developed for mA methylome, mA-related enzymes (writers and erasers), as well as its functions in mRNA and tRNA.
Adenosine ; analogs & derivatives ; chemistry ; DNA Methylation ; Epigenomics ; Gene Expression Regulation ; Humans ; RNA, Messenger ; chemistry ; RNA, Transfer ; chemistry

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

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

Mitochondrial tRNAgene mutation is closely related to acoustic nerve deafness. Some mutations can affect the structure and transcriptional processing of tRNA, for instance m.7444G>A mutation in tRNAprecursor 3' side, m.7472 insC as well as m.7511T>C mutations in the stem and ring of tRNA, may influence tRNAstability, thus affect the synthesis of mitochondrial peptides, reduce the production of ATP and cause deafness. This article focuses on mitochondrial tRNAgene mutations as well as the mechanism underlying hearing loss.
Amino Acid Sequence ; Base Sequence ; Genetic Predisposition to Disease ; genetics ; Hearing Loss ; genetics ; Humans ; Mitochondrial Proteins ; biosynthesis ; genetics ; Mutation ; Nucleic Acid Conformation ; RNA ; chemistry ; genetics ; RNA, Transfer, Ser ; chemistry ; genetics

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation

OBJECTIVETo study the relationship between mitochondrial DNA (mtDNA) mutations and hypertension.

METHODSClinical data of two pedigrees with maternally transmitted hypertension was collected. Whole mtDNA sequence was analyzed.

RESULTSThe family members on the maternal side presented with various levels of hypertension, with the onset age ranging from 44 to 55 years old. Analysis of the mtDNA sequence of the two families members showed all patients have carried a matrilineal 4329C> G mutation of the tRNA(Ile) and tRNA(Gln) genes. The same mutation was not found in 366 healthy controls. The 4329C site of mtDNA is highly conserved across species, and has been associated with the fidelity of amino acid accept arm of the tRNAs, as well as functionality and stability in the formation of tRNAs.

CONCLUSIONThe 4329C> G point mutation in tRNA(Ile) and tRNA(Gln) probably has contributed to the pathogenesis of hypertension, possibly in association with other modifying factors.

Adult ; Base Sequence ; DNA Mutational Analysis ; DNA, Mitochondrial ; chemistry ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Hypertension ; genetics ; Male ; Middle Aged ; Molecular Sequence Data ; Pedigree ; Point Mutation ; RNA, Transfer, Gln ; genetics ; RNA, Transfer, Ile ; genetics ; Sequence Homology, Amino Acid

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

The arginyl-tRNA synthetase (ArgRS) catalyzes the esterification reaction between L-arginine and its cognate tRNA(Arg). Previously reported structures of ArgRS shed considerable light on the tRNA recognition mechanism, while the aspect of amino acid binding in ArgRS remains largely unexplored. Here we report the first crystal structure of E. coli ArgRS (eArgRS) complexed with L-arginine, and a series of mutational studies using isothermal titration calorimetry (ITC). Combined with previously reported work on ArgRS, our results elucidated the structural and functional roles of a series of important residues in the active site, which furthered our understanding of this unique enzyme.
Arginine ; chemistry ; Arginine-tRNA Ligase ; chemistry ; Binding Sites ; Catalytic Domain ; Crystallography, X-Ray ; Escherichia coli ; Ligands ; Mutagenesis, Site-Directed ; Protein Binding ; Protein Conformation ; RNA, Transfer ; chemistry ; Structure-Activity Relationship