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.     

Novel Roles of RNA m6A Methylation Regulators in the Occurrence of Alzheimer’s Disease and the Subtype Classification

Alzheimer’s disease (AD) is one of the most common forms of dementia, closely related to epigenetic factors. N6-methyladenosine (m6A) is the most abundant RNA modification, affecting the pathogenesis and development of neurodegenerative diseases. This study was the first exploration of the combined role of 25 common m6A RNA methylation regulators in AD through the integrated bioinformatics approaches. The 14 m6A regulators related to AD were selected by analyzing differences between AD patients and normal controls. Based on the selected m6A regulators, AD patients could be well classified into two m6A models using consensus clustering. The two clusters of patients had different immune profiles, and m6A regulators were associated with the components of immune cells. Additionally, there were 19 key AD genes obtained by screening differential genes through weighted gene co-expression network and least absolute shrinkage and selection operator regression analysis, which were highly associated with important m6A regulators during the occurrence of AD. More interestingly, NOTCH2 and NME1 could be potential targets for m6A regulation of AD. Taken together, these findings indicate that dysregulation of m6A methylation affects the occurrence of AD and is vital for the subtype classification and immune infiltration of AD.     

Expression and Prognostic Characteristics of m6A RNA Methylation Regulators in Colon Cancer

Colon cancer is a common and leading cause of death and malignancy worldwide. N6-methylation of adenosine (m6A) is the most common reversible mRNA modification in eukaryotes, and it plays a crucial role in various biological functions in vivo. Dysregulated expression and genetic changes of m6A regulators have been correlated with tumorigenesis, cancer cell proliferation, tumor microenvironment, and prognosis in cancers. This study used RNA-seq and colon cancer clinical data to explore the relationship between N6-methylation and colon cancer. Based on the seven m6A regulators related to prognosis, three molecular subgroups of colon cancer were identified. Surprisingly, we found that each subgroup had unique survival characteristics. We then identified three subtypes of tumors based on 299 m6A phenotype-related genes, and one subtype was characterized as an immunosuppressive tumor and patients in this subtype may be more suitable for immunotherapy than other subtypes. Finally, using m6A-related genes and clinical information from The Cancer Genome Atlas cohort, we constructed a prognosis model, and this model could be used to predict the prognosis of patients in clinics.     

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.     

Effects of In Ovo Administration of DHEA on Lipid Metabolism and Hepatic Lipogenetic Genes Expression in Broiler Chickens During Embryonic Development

In order to study the mechanism of DHEA (Dehydroepiandrosterone) in reducing fat in broiler chickens during embryonic development, fertilized eggs were administrated with DHEA before incubation and its effect on lipid metabolism and expression of hepatic lipogenetic genes was investigated. The mRNA levels of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), apolipoprotein B100 (apoB100) and sterol regulator element binding protein-1c (SREBP-1c) were determined using real time quantitative PCR. Samples of livers were collected from the chickens on days 9, 14, and 19 of embryonic development as well as at hatching. Blood samples were extracted on days 14, 19 of incubation and at hatching. The results showed that DHEA decreased the concentration of triacyglycerol in the blood and the content in liver, and the mRNA levels of ACC, FAS, ME, SREBP-1c and apoB. This suggested that DHEA decreased the expression of hepatic lipogenetic genes and suppressed triglycerols transport, by which it reduced the deposition of fat in adipose tissue in broiler chickens during embryonic development and hatching.     

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.     

The m6A Methyltransferase METTL3-Mediated N6-Methyladenosine Modification of DEK mRNA to Promote Gastric Cancer Cell Growth and Metastasis

Gastric cancer (GC) is the fifth most common cancer and the third deadliest cancer in the world, and the occurrence and development of GC are influenced by epigenetics. Methyltransferase-like 3 (METTL3) is a prominent RNA n6-adenosine methyltransferase (m6A) that plays an important role in tumor growth by controlling the work of RNA. This study aimed to reveal the biological function and molecular mechanism of METTL3 in GC. The expression level of METTL3 in GC tissues and cells was detected by qPCR, Western blot and immunohistochemistry, and the expression level and prognosis of METTL3 were predicted in public databases. CCK-8, colony formation, transwell and wound healing assays were used to study the effect of METTL3 on GC cell proliferation and migration. In addition, the enrichment effect of METTL3 on DEK mRNA was detected by the RIP experiment, the m6A modification effect of METTL3 on DEK was verified by the MeRIP experiment and the mRNA half-life of DEK when METTL3 was overexpressed was detected. The dot blot assay detects m6A modification at the mRNA level. The effect of METTL3 on cell migration ability in vivo was examined by tail vein injection of luciferase-labeled cells. The experimental results showed that METTL3 was highly expressed in GC tissues and cells, and the high expression of METTL3 was associated with a poor prognosis. In addition, the m6A modification level of mRNA was higher in GC tissues and GC cell lines. Overexpression of METTL3 in MGC80-3 cells and AGS promoted cell proliferation and migration, while the knockdown of METTL3 inhibited cell proliferation and migration. The results of in vitro rescue experiments showed that the knockdown of DEK reversed the promoting effects of METTL3 on cell proliferation and migration. In vivo experiments showed that the knockdown of DEK reversed the increase in lung metastases caused by the overexpression of METTL3 in mice. Mechanistically, the results of the RIP experiment showed that METTL3 could enrich DEK mRNA, and the results of the MePIP and RNA half-life experiments indicated that METTL3 binds to the 3’UTR of DEK, participates in the m6A modification of DEK and promotes the stability of DEK mRNA. Ultimately, we concluded that METTL3 promotes GC cell proliferation and migration by stabilizing DEK mRNA expression. Therefore, METTL3 is a potential biomarker for GC prognosis and a therapeutic target.     

Small-Molecule Inhibitors of the RNA M6A Demethylases FTO Potently Support the Survival of Dopamine Neurons

The fat mass and obesity-associated protein (FTO), an RNA N⁶-methyladenosine (m⁶A) demethylase, is an important regulator of central nervous system development, neuronal signaling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated K_d = 185 nM; IC₅₀ = 1.46 µM (compound 2) and K_d = 337 nM; IC₅₀ = 28.9 µM (compound 3). Importantly, the treatment of mouse midbrain dopaminergic neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, both the best inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The FTO inhibitors demonstrated increased potency as compared to our recently developed ALKBH5 m⁶A demethylase inhibitors in protecting dopamine neurons. Inhibition of m⁶A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m⁶A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases.     

The Impact of m1A Methylation Modification Patterns on Tumor Immune Microenvironment and Prognosis in Oral Squamous Cell Carcinoma

N1-methyladenosine (m1A) modification widely participates in the occurrence and progression of numerous diseases. Nevertheless, the potential roles of m1A in the tumor immune microenvironment (TIME) are still not fully understood. Based on 10 m1A methylation regulators, we comprehensively explored the m1A modification patterns in 502 patients with oral squamous cell carcinoma (OSCC). The m1A modification patterns were correlated with TIME characteristics and the m1A score was established to evaluate the effect of the m1A modification patterns on individual OSCC patients. The TIME characteristics and survival outcomes under the three m1A modification patterns were significantly distinct. OSCC patients in the high m1A score group were characterized by poorer prognosis, lower immune infiltration, lower ssGSEA score, lower expression levels of immune checkpoint molecules, and higher tumor mutation loads. The present study revealed that m1A modification might be associated with the TIME in OSCC, and has potential predictive ability for the prognosis of OSCC.     

Black Raspberry Seed Oil Improves Lipid Metabolism by Inhibiting Lipogenesis and Promoting Fatty‐Acid Oxidation in High‐Fat Diet‐Induced Obese Mice and db/db Mice

The objective of this study was to evaluate the beneficial effect of α‐linolenic acid‐rich black raspberry seed (BRS) oil on lipid metabolism in high‐fat diet (HFD)‐induced obese and db/db mice. Five‐week‐old C57BL/6 mice were fed diets consisting of 50% calories from lard, 5% from soybean, and 5% from corn oil (HFD), or 50% calories from lard and 10% from BRS oil (HFD + BRS oil diet) for 12 weeks. Six‐week‐old C57BL/KsJ‐db/db mice were fed diets consisting of 16% calories from soybean oil (standard diet), 8% from soybean, and 8% from BRS oil, or 16% from BRS oil for 10 weeks. The BRS oil diets lowered the levels of triacylglycerol, nonesterified fatty acids, and total cholesterol in serum and liver of both of the obese and db/db mice as compared with the HFD and standard diet, respectively. mRNA levels of lipogenesis markers including cluster of differentiation 36, fatty‐acid‐binding protein 1, sterol regulatory element binding protein 1c, fatty‐acid synthase, and solute carrier family 25 member 1 in the liver of the BRS oil groups were lower than those in the liver of the HFD and standard groups in the obese and db/db mice, respectively. On the other hand, fatty‐acid oxidation markers including carnitine palmitoyltransferase 1A, acyl‐CoA dehydrogenase, hydroxylacyl‐CoA dehydrogenase α, and acyl‐CoA oxidase in the liver of the BRS oil groups were higher than those in the liver of the HFD and standard groups in the obese and db/db mice, respectively. Peroxisome proliferator‐activated receptor α mRNA and protein levels increased in the liver and epididymal adipose tissue of the obese and db/db mice fed BRS oil compared with HFD and standard diet, respectively. BRS oil might improve lipid metabolism by inhibiting lipogenesis and promoting fatty‐acid oxidation in HFD‐induced obese and db/db mice.     

Site‐Specific Modification of the 6‐Amino Group of Adenosine in RNA by an Interstrand Functionality‐Transfer Reaction With an S‐Functionalized 4‐Thiothymidine

Non‐natural RNA modifications have been widely used to study the function and structure of RNA. Expanding the study of RNA further requires versatile and efficient tools for site‐specific RNA modification. We recently established a new strategy for the site‐specific modification of RNA based on a functionality‐transfer reaction between an oligodeoxynucleotide (ODN) probe and an RNA substrate. 2′‐Deoxy‐6‐thioguanosine was used to anchor the transfer group, and the 4‐amino group of cytosine or the 2‐amino group of guanine was specifically modified. In this study, 2′‐deoxy‐4‐thiothymidine was adopted as a new platform to target the 6‐amino group of adenosine. The (E)‐pyridinyl vinyl keto transfer group was attached to the 4‐thioT in the ODN probe, and it was efficiently and specifically transferred to the 6‐amino group of the opposing adenosine in RNA in the presence of CuCl₂. This method expands the available RNA target sites for specific modification.     

Short‐Chain Fatty Acids Enhance the Lipid Accumulation of 3T3‐L1 Cells by Modulating the Expression of Enzymes of Fatty Acid Metabolism

Short‐chain fatty acids (SCFA) such as acetic acid, propionic acid, and butyric acid are produced by fermentation by gut microbiota. In this paper, we investigate the effects of SCFA on 3T3‐L1 cells and the underlying molecular mechanisms. The cells were treated with acetic acid, propionic acid, or butyric acid when cells were induced to differentiate into adipocytes. MTT assay was employed to detect the viability of 3T3‐L1 cells. Oil Red O staining was used to visualize the lipid content in 3T3‐L1 cells. A triglyceride assay kit was used to detect the triacylglycerol content in 3T3‐L1 cells. qRT‐PCR and Western blot were used to evaluate the expression of metabolic enzymes. MTT results showed that safe concentrations of acetic acid, propionic acid, and butyric acid were less than 6.4, 3.2, and 0.8 mM, respectively. Oil Red O staining and triacylglycerols detection results showed that treatment with acetic acid, propionic acid, and butyric acid accelerated the 3T3‐L1 adipocyte differentiation. qRT‐PCR and Western blot results showed that the expressions of lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4), fatty acid transporter protein 4 (FATP4), and fatty acid synthase (FAS) were significantly increased by acetic acid, propionic acid, and butyric acid treatment during adipose differentiation (p < 0.05). In conclusion, SCFA promoted lipid accumulation by modulating the expression of enzymes of fatty acid metabolism.     

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.