Genome-Wide Identification,Classification and Expression Analysis of m6A Gene Family in Solanum lycopersicum

Advanced knowledge of messenger RNA (mRNA) N⁶-methyladenosine (m⁶A) and DNA N⁶-methyldeoxyadenosine (6 mA) redefine our understanding of these epigenetic modifications. Both m⁶A and 6mA carry important information for gene regulation, and the corresponding catalytic enzymes sometimes belong to the same gene family and need to be distinguished. However, a comprehensive analysis of the m⁶A gene family in tomato remains obscure. Here, 24 putative m⁶A genes and their family genes in tomato were identified and renamed according to BLASTP and phylogenetic analysis. Chromosomal location, synteny, phylogenetic, and structural analyses were performed, unravelling distinct evolutionary relationships between the MT-A70, ALKBH, and YTH protein families, respectively. Most of the 24 genes had extensive tissue expression, and 9 genes could be clustered in a similar expression trend. Besides, SlYTH1 and SlYTH3A showed a different expression pattern in leaf and fruit development. Additionally, qPCR data revealed the expression variation under multiple abiotic stresses, and LC-MS/MS determination exhibited that the cold stress decreased the level of N⁶ 2′-O dimethyladenosine (m⁶Am). Notably, the orthologs of newly identified single-strand DNA (ssDNA) 6mA writer–eraser–reader also existed in the tomato genome. Our study provides comprehensive information on m⁶A components and their family proteins in tomato and will facilitate further functional analysis of the tomato N⁶-methyladenosine modification genes.     

m6A Modified Short RNA Fragments Inhibit Cytoplasmic TLS/FUS Aggregation Induced by Hyperosmotic Stress

Translocated in LipoSarcoma/Fused in Sarcoma (TLS/FUS) is a nuclear RNA binding protein whose mutations cause amyotrophic lateral sclerosis. TLS/FUS undergoes LLPS and forms membraneless particles with other proteins and nucleic acids. Interaction with RNA alters conformation of TLS/FUS, which affects binding with proteins, but the effect of m⁶A RNA modification on the TLS/FUS–RNA interaction remains elusive. Here, we investigated the binding specificity of TLS/FUS to m⁶A RNA fragments by RNA pull down assay, and elucidated that both wild type and ALS-related TLS/FUS mutants strongly bound to m⁶A modified RNAs. TLS/FUS formed cytoplasmic foci by treating hyperosmotic stress, but the cells transfected with m⁶A-modified RNAs had a smaller number of foci. Moreover, m⁶A-modified RNA transfection resulted in the cells obtaining higher resistance to the stress. In summary, we propose TLS/FUS as a novel candidate of m⁶A recognition protein, and m⁶A-modified RNA fragments diffuse cytoplasmic TLS/FUS foci and thereby enhance cell viability.     

m6A-Mediated PPARA Translational Suppression Contributes to Corticosterone-Induced Visceral Fat Deposition in Chickens

Excess fat deposition in broilers leads to great economic losses and is harmful to consumers’ health. Chronic stress in the life cycle of chickens could be an important trigger. However, the underlying mechanisms are still unclear. In this study, 30-day-old chickens were subcutaneously injected with 2 mg/kg corticosterone (CORT) twice a day for 14 days to simulate long-term stress. It was shown that chronic CORT exposure significantly increased plasma triglyceride concentrations and enlarged the adipocyte sizes in chickens. Meanwhile, chronic CORT administration significantly enlarged the adipocyte sizes, increased the protein contents of FASN and decreased HSL, ATGL, Beclin1 and PPARA protein levels. Moreover, global m⁶A methylations were significantly reduced and accompanied by downregulated METTL3 and YTHDF2 protein expression by CORT treatment. Interestingly, the significant differences of site-specific m⁶A demethylation were observed in exon7 of PPARA mRNA. Additionally, a mutation of the m⁶A site in the PPARA gene fused GFP and revealed that demethylated RRACH in PPARA CDS impaired protein translation in vitro. In conclusion, these results indicated that m⁶A-mediated PPARA translational suppression contributes to CORT-induced visceral fat deposition in chickens, which may provide a new target for the treatment of Cushing’s syndrome.     

Comprehensive Analysis of Long Noncoding RNA Modified by m6A Methylation in Oxidative and Glycolytic Skeletal Muscles

N⁶-methyladenosine (m⁶A) is the most common modification in eukaryotic RNAs. Accumulating evidence shows m⁶A methylation plays vital roles in various biological processes, including muscle and fat differentiation. However, there is a lack of research on lncRNAs’ m⁶A modification in regulating pig muscle-fiber-type conversion. In this study, we identified novel and differentially expressed lncRNAs in oxidative and glycolytic skeletal muscles through RNA-seq, and further reported the m⁶A-methylation patterns of lncRNAs via MeRIP-seq. We found that most lncRNAs have one m⁶A peak, and the m⁶A peaks were preferentially enriched in the last exon of the lncRNAs. Interestingly, we found that lncRNAs’ m⁶A levels were positively correlated with their expression homeostasis and levels. Furthermore, we performed conjoint analysis of MeRIP-seq and RNA-seq data and obtained 305 differentially expressed and differentially m⁶A-modified lncRNAs (dme-lncRNAs). Through QTL enrichment analysis of dme-lncRNAs and PPI analysis for their cis-genes, we finally identified seven key m⁶A-modified lncRNAs that may play a potential role in muscle-fiber-type conversion. Notably, inhibition of one of the key lncRNAs, MSTRG.14200.1, delayed satellite cell differentiation and stimulated fast-to-slow muscle-fiber conversion. Our study comprehensively analyzed m⁶A modifications on lncRNAs in oxidative and glycolytic skeletal muscles and provided new targets for the study of pig muscle-fiber-type conversion.     

Circular RNA,the Key for Translation

It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m⁶A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5′ end-independent translation initiation. Today a new family of so-called “noncoding” circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5′ end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3′–5′ interaction have been reported, including m⁶A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and noncovalently closed circular mRNA translation landscape shows that RNA with circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.     

Leptin Reduces Plin5 m6A Methylation through FTO to Regulate Lipolysis in Piglets

Perilipin5 (Plin5) is a scaffold protein that plays an important role in lipid droplets (LD) formation, but the regulatory effect of leptin on it is unclear. Our study aimed to explore the underlying mechanisms by which leptin reduces the N⁶-methyladenosine (m⁶A) methylation of Plin5 through fat mass and obesity associated genes (FTO) and regulates the lipolysis. To this end, 24 Landrace male piglets (7.73 ± 0.38 kg) were randomly sorted into two groups, either a control group (Control, n = 12) or a 1 mg/kg leptin recombinant protein treatment group (Leptin, n = 12). After 4 weeks of treatment, the results showed that leptin treatment group had lower body weight, body fat percentage and blood lipid levels, but the levels of Plin5 mRNA and protein increased significantly in adipose tissue (p < 0.05). Leptin promotes the up-regulation of FTO expression level in vitro, which in turn leads to the decrease of Plin5 M⁶A methylation (p < 0.05). In in vitro porcine adipocytes, overexpression of FTO aggravated the decrease of M⁶A methylation and increased the expression of Plin5 protein, while the interference fragment of FTO reversed the decrease of m⁶A methylation (p < 0.05). Finally, the overexpression in vitro of Plin5 significantly reduces the size of LD, promotes the metabolism of triglycerides and the operation of the mitochondrial respiratory chain, and increases thermogenesis. This study clarified that leptin can regulate Plin5 M⁶A methylation by promoting FTO to affect the lipid metabolism and energy consumption, providing a theoretical basis for treating diseases related to obesity.     

Glucocorticoid Regulates the Synthesis of Porcine Muscle Protein through m6A Modified Amino Acid Transporter SLC7A7

The occurrence of stress is unavoidable in the process of livestock production, and prolonged stress will cause the decrease of livestock productivity. The stress response is mainly regulated by the hypothalamic-pituitary-adrenal axis (HPA axis), which produces a large amount of stress hormones, namely glucocorticoids (GCs), and generates a severe impact on the energy metabolism of the animal body. It is reported that m⁶A modification plays an important role in the regulation of stress response and also participates in the process of muscle growth and development. In this study, we explored the effect of GCs on the protein synthesis procession of porcine skeletal muscle cells (PSCs). We prove that dexamethasone affects the expression of SLC7A7, a main amino acid transporter for protein synthesis by affecting the level of m⁶A modification in PSCs. In addition, we find that SLC7A7 affects the level of PSC protein synthesis by regulating the conduction of the mTOR signaling pathway, which indicates that the reduction of SLC7A7 expression may alleviate the level of protein synthesis under stress conditions.     

Reduction of Methyltransferase-like 3-Mediated RNA N6-Methyladenosine Exacerbates the Development of Psoriasis Vulgaris in Imiquimod-Induced Psoriasis-like Mouse Model

N6-methyladenosine (m⁶A) methylation is the most pervasive and intensively studied mRNA modification, which regulates gene expression in different physiological processes, such as cell proliferation, differentiation, and inflammation. Studies of aberrant m⁶A in human diseases such as cancer, obesity, infertility, neuronal disorders, immune diseases, and inflammation are rapidly evolving. However, the regulatory mechanism and physiological significance of m⁶A methylation in psoriasis vulgaris are still poorly understood. In this study, we found that m⁶A methylation and Methyltransferase-like 3 (METTL3) were both downregulated in psoriatic skin lesions and were negatively correlated with Psoriasis Area and Severity Index (PASI) scores. Inhibiting m⁶A methylation by knocking down Mettl3 promoted the development of psoriasis and increased its severity in imiquimod-induced psoriasis-like model mice. Our results indicate a critical role of METTL3- mediated m⁶A methylation in the pathogenesis of psoriasis vulgaris.     

Emerging Mutual Regulatory Roles between m6A Modification and microRNAs

N⁶-metyladenosine (m⁶A), one of the most common RNA methylation modifications in mammals, has attracted extensive attentions owing to its regulatory roles in a variety of physiological and pathological processes. As a reversible epigenetic modification on RNAs, m⁶A is dynamically mediated by the functional interplay among the regulatory proteins of methyltransferases, demethylases and methyl-binding proteins. In recent years, it has become increasingly clear that m⁶A modification is associated with the production and function of microRNAs (miRNAs). In this review, we summarize the specific kinds of m⁶A modification methyltransferases, demethylases and methyl-binding proteins. In particular, we focus on describing the roles of m⁶A modification and its regulatory proteins in the production and function of miRNAs in a variety of pathological and physiological processes. More importantly, we further discuss the mediating mechanisms of miRNAs in m⁶A modification and its regulatory proteins during the occurrence and development of various diseases.