There are a variety of post-transcriptional modifications in mRNA, which regulate the stability, splicing, translation, transport and other processes of mRNA, followed by affecting cell development, body immunity, learning and cognition and other important physiological functions. m⁶A modification is one of the most abundant post-transcriptional modifications widely existing in mRNA, regulating the metabolic activities of RNA and affecting gene expression. m⁶A modified homeostasis is critical for the development and maintenance of the nervous system. In recent years, m⁶A modification has been found in neurodegenerative diseases, mental diseases and brain tumors. This review summarizes the role of m⁶A methylation modification in the development, function and related diseases of the central nervous system in recent years, providing potential clinical therapeutic targets for neurological diseases.
Methylation ; Central Nervous System/metabolism* ; RNA, Messenger/metabolism* ; RNA

Objective To investigate the molecular mechanism of taurine regulating the polarization of M2 macrophages by mitophagy. Methods THP-1 cells were divided into four groups: M0 group (THP-1 cells were treated by 100 nmol/L phorbol myristate ester for 48 hours to polarize into M0), M2 group (THP-1 cells were induced to polarize into M2 macrophages by 20 ng/mL interferon-4 (IL-4) for 48 hours), M2 combined with taurine groups (added with 40 or 80 mmol/L taurine on the basis of M2 macrophages). The mRNA expression of mannose receptor C type 1(MRC-1), C-C motif chemokine ligand 22(CCL22) and dendritic cell-specific ICAM-3 grabbing non-integrin (CD209) in M2 macrophages were detected by quantitative real-time PCR. Mitochondrial and lysosome probes were used to detect the number of mitochondria and lysosomes by multifunction microplate reader and confocal laser scanning microscope. The level of mitochondrial membrane potential (MMP) was detected by JC-1 MMP assay kit. The expression of mitophagy-related proteins PTEN-induced putative kinase 1 (PINK1) and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blot analysis. Results Compared with M0 group, the expression of MRC-1, CCL22, CD209 and PINK1, the number of mitochondria and the level of MMP in M2 group were significantly increased, whereas the number of lysosomes and LC3II/LC3I ratio were decreased. Compared with M2 group, the expressions of MRC-1, CCL22 and CD209, the number of mitochondria and the level of MMP in M2 combined with taurine group dropped significantly while the number of lysosomes was found increased, and the protein expression of PINK1 and LC3II/LC3I ratio were also increased. Conclusions The polarization of M2 macrophages is regulated by taurine to prevent excessive polarization via reducing the level of MMP, improving the level of mitophagy, reducing the number of mitochondria, and inhibiting the mRNA expression of polarization markers in M2 macrophages.
Mitophagy ; Taurine ; Macrophages/metabolism* ; Protein Kinases/metabolism* ; RNA, Messenger

We have yet to develop a fundamental understanding of the molecular complexities of human spermatozoa. This encompasses the unique packaging and structure of the sperm genome along with their paternally derived RNAs in preparation for their delivery to the egg. The diversity of these transcripts is vast, including several anti-sense molecules resembling known regulatory micro-RNAs. The field is still grasping with its delivery to the oocyte at fertilization and possible significance. It remains tempting to analogize them to maternally-derived transcripts active in early embryo patterning. Irrespective of their role in the embryo, their use as a means to assess male factor infertility is promising.
DNA ; genetics ; metabolism ; Humans ; Male ; RNA, Messenger ; genetics ; Spermatozoa ; metabolism

Alternative splicing (AS) is an evolutionarily conserved mechanism that removes introns and ligates exons to generate mature messenger RNAs (mRNAs), extremely improving the richness of transcriptome and proteome. Both mammal hosts and pathogens require AS to maintain their life activities, and inherent physiological heterogeneity between mammals and pathogens makes them adopt different ways to perform AS. Mammals and fungi conduct a two-step transesterification reaction by spliceosomes to splice each individual mRNA (named cis -splicing). Parasites also use spliceosomes to splice, but this splicing can occur among different mRNAs (named trans -splicing). Bacteria and viruses directly hijack the host's splicing machinery to accomplish this process. Infection-related changes are reflected in the spliceosome behaviors and the characteristics of various splicing regulators (abundance, modification, distribution, movement speed, and conformation), which further radiate to alterations in the global splicing profiles. Genes with splicing changes are enriched in immune-, growth-, or metabolism-related pathways, highlighting approaches through which hosts crosstalk with pathogens. Based on these infection-specific regulators or AS events, several targeted agents have been developed to fight against pathogens. Here, we summarized recent findings in the field of infection-related splicing, including splicing mechanisms of pathogens and hosts, splicing regulation and aberrant AS events, as well as emerging targeted drugs. We aimed to systemically decode host-pathogen interactions from a perspective of splicing. We further discussed the current strategies of drug development, detection methods, analysis algorithms, and database construction, facilitating the annotation of infection-related splicing and the integration of AS with disease phenotype.
Animals ; Alternative Splicing/genetics* ; RNA Splicing ; Spliceosomes/metabolism* ; RNA, Messenger/metabolism* ; Communicable Diseases/genetics* ; Mammals/metabolism*

METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m6A methyltransferase complex (MTC) that installs m6A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell (mESC). While comparable global hypo-methylation in RNA m6A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m6A-independent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification. Mechanistically, METTL14, but not METTL3, binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression. The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs. This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m6A, and critically impacts transcriptional regulation, stemness maintenance, and differentiation of mESCs.
Animals ; Mice ; Methylation ; Chromatin ; Histones/metabolism* ; RNA, Messenger/genetics* ; Methyltransferases/metabolism* ; RNA/metabolism*

Parasite-derived non-coding RNAs (ncRNAs) not only contribute to life activities of parasites, and microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) may generate a competitive endogenous RNA (ceRNA) regulatory network with host miRNAs and mRNAs via extracellular vesicles, thereby participating in infection and pathogenic processes. This article presents an overview of characterizing ncRNAs derived from parasites and the cross-species regulatory role of parasite-derived ncRNAs in host gene expression and its underlying mechanisms.
Animals ; Parasites ; Gene Regulatory Networks ; MicroRNAs/metabolism* ; RNA, Messenger/genetics* ; RNA, Circular/genetics* ; RNA, Competitive Endogenous

Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.
Female ; Humans ; Pregnancy ; MicroRNAs/metabolism* ; RNA, Circular/metabolism* ; RNA, Messenger/metabolism* ; Pre-Eclampsia/metabolism* ; Placenta/metabolism* ; RNA/metabolism* ; RNA, Small Interfering ; Gene Expression Profiling

Animals ; Biological Evolution ; Fungi ; genetics ; Plants ; genetics ; RNA, Messenger ; metabolism

To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.
Gene Library ; Humans ; RNA, Messenger ; analysis ; U937 Cells ; metabolism

OBJECTIVE: To investigate the molecular mechanism in stable cell strains expressing Mini-hF9 gene with nonsense mutation. METHODS: Mini-hF9 gene and its nonsense mutants were transfected into HeLa cells independently, and stable cell strains were obtained after G418 resistance screening and monoclonal transformation. The altered splicing and protein expression of mRNA in Mini-hF9 gene in stable cell strains were detected by using RT-PCR and Western blot. RESULTS: The wild type and nonsense mutated human coagulation factor IX stable cell strains were constructed successfully, which were named HeLa-F9-WT, HeLa-F9-M1 and HeLa-F9-M2. Only normal splicing Norm was detected in the wild-type cell strain HeLa-F9-WT; Norm and Alt-S1 splicing were detected in HeLa-F9-M1; while Norm, Alt-S1 and Alt-S2 splicing were detected in HeLa-F9-M2. CONCLUSION The nonsense associated altered splicing (NAS) pathway, which generated alternately spliced transcripts, might be triggered in coagulation factor IX gene with nonsense mutation.
Codon, Nonsense ; Factor IX/metabolism* ; HeLa Cells ; Humans ; Mutation ; RNA Splicing ; RNA, Messenger/metabolism*