Wilms' Tumor 1-associating Protein Research Articles

HIF1α-mediated transactivation of WTAP promotes AML cell proliferation via m6A-dependent stabilization of KDM4B mRNA.

Hypoxia inducible factor 1α (HIF1α) is abnormally overexpressed in t(8;21) acute myeloid leukemia (AML) and functions as an oncogene through transactivating DNA methyltransferase 3 alpha leading to DNA hypermethylation. However, it remains unclear whether HIF1α influences RNA N6-methyladenosine (m6A) methyltransferases. Here, we show that HIF1α promotes the expression of Wilms tumor 1-associated protein (WTAP), a main component of the m6A methyltransferase complex, markedly alters the transcriptome-wide m6A distribution and enhances cell proliferation in t(8;21) AML. In agreement with this, WTAP is overexpressed and predicts poor prognosis in t(8;21) AML patients. Moreover, WTAP knockdown inhibits growth, and induces apoptosis and differentiation of leukemia cells. Mechanistically, HIF1α transactivates WTAP gene expression by directly binding to the hypoxia-response element of its promoter region. Pharmacological or genetic intervention in the HIF1α-WTAP axis results in the reduction of m6A level on lysine demethylase 4B (KDM4B) transcripts and increased its degradation, correlated with lower expression of KDM4B and higher trimethylation levels of histone H3 on lysine 9. KDM4B knockdown inhibits leukemia cell growth in vitro and in mice. Thus, HIF1α-mediated WTAP high expression enhances the malignant behavior of leukemia cells and drives a crosstalk between m6A RNA methylation and histone methylation through monitoring m6A-dependant KDM4B translation.

Development and validation of an m6A RNA methylation regulator-based signature for the prediction of prognosis and immunotherapy in cutaneous melanoma.

Development and validation of an m6A RNA methylation regulator-based signature for the prediction of prognosis and immunotherapy in cutaneous melanoma.

WTAP-Mediated m6A RNA Methylation Regulates the Differentiation of Bone Marrow Mesenchymal Stem Cells via the miR-29b-3p/HDAC4 Axis.

N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the progression of osteoporosis (OP), providing novel insights into the pathogenesis of OP. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been studied in OP. Here we explored the biological role and underlying mechanism of WTAP in OP and the differentiation of bone marrow mesenchymal stem cells (BMMSCs). We demonstrated that WTAP was expressed at low levels in bone specimens from patients with OP and OVX mice. Functionally, WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs in vitro and in vivo. In addition, microRNA-29b-3p (miR-29b-3p) was identified as a downstream target of WTAP. M6A modifications regulated by WTAP led to increased miR-29b-3p expression. WTAP interacted with the microprocessor protein DGCR8 and accelerated the maturation of pri-miR-29b-3p in an m6A-dependent manner. Target prediction and dual-luciferase reporter assays identified the direct binding sites of miR-29b-3p with histone deacetylase 4 (HDAC4). WTAP-mediated m6A modification promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs through the miR-29b-3p/HDAC4 axis. Furthermore, WTAP-mediated m6A methylation negatively regulates osteoclast differentiation. Collectively, our study first identified a critical role of WTAP-mediated m6A methylation in BMMSC differentiation and highlighted WTAP as a potential therapeutic target for OP treatment.

Exosome-derived circCCAR1 promotes CD8 + T-cell dysfunction and anti-PD1 resistance in hepatocellular carcinoma

BackgroundCircular RNAs (circRNAs) can be encapsulated into exosomes to participate in intercellular communication, affecting the malignant progression of a variety of tumors. Dysfunction of CD8 + T cells is the main factor in immune escape from hepatocellular carcinoma (HCC). Nevertheless, the effect of exosome-derived circRNAs on CD8 + T-cell dysfunction needs further exploration.MethodsThe effect of circCCAR1 on the tumorigenesis and metastasis of HCC was assessed by in vitro and in vivo functional experiments. The function of circCCAR1 in CD8 + T-cell dysfunction was measured by enzyme-linked immunosorbent assay (ELISA), western blotting and flow cytometry. Chromatin immunoprecipitation, biotinylated RNA pull-down, RNA immunoprecipitation, and MS2 pull-down assays were used to the exploration of mechanism. A mouse model with reconstituted human immune system components (huNSG mice) was constructed to explore the role of exosomal circCCAR1 in the resistance to anti-PD1 therapy in HCC.ResultsIncreased circCCAR1 levels existed in tumor tissues and exosomes in the plasma of HCC patients, in the culture supernatant and HCC cells. CircCCAR1 accelerated the growth and metastasis of HCC in vitro and in vivo. E1A binding protein p300 (EP300) and eukaryotic translation initiation factor 4A3 (EIF4A3) promoted the biogenesis of circCCAR1, and Wilms tumor 1-associated protein (WTAP)-mediated m6A modification enhanced circCCAR1 stability by binding insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). CircCCAR1 acted as a sponge for miR-127-5p to upregulate its target WTAP and a feedback loop comprising circCCAR1/miR-127-5p/WTAP axis was formed. CircCCAR1 is secreted by HCC cells in a heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1)-dependent manner. Exosomal circCCAR1 was taken in by CD8 + T cells and caused dysfunction of CD8 + T cells by stabilizing the PD-1 protein. CircCCAR1 promoted resistance to anti-PD1 immunotherapy. Furthermore, increased cell division cycle and apoptosis regulator 1 (CCAR1) induced by EP300 promoted the binding of CCAR1 and β-catenin protein, which further enhanced the transcription of PD-L1.ConclusionsThe circCCAR1/miR-127-5p/WTAP feedback loop enhances the growth and metastasis of HCC. Exosomal circCCAR1 released by HCC cells contributes to immunosuppression by facilitating CD8 + T-cell dysfunction in HCC. CircCCAR1 induces resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for HCC patients.

Caprin-1 plays a role in cell proliferation and Warburg metabolism of esophageal carcinoma by regulating METTL3 and WTAP

BackgroundCytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is implicated in cancer cell proliferation and tumorigenesis; however, its role in the development of esophageal carcinoma (ESCA) has not been examined.MethodsBiological methods and data analysis were used to investigate the expression of Caprin-1 in ESCA tissue and cell lines. We comprehensively analyzed the mRNA expression and prognostic values, signalling pathways of CAPRIN1 in ESCA using public databases online. Biological functions of CAPRIN1 were performed by clorimetric growth assay, EdU staining, colony formation, flow cytometry, apoptosis analysis, Western blot, lactate detection assay, extracellular acidification rates. The underlying mechanism was determined via flow cytometric analysis, Western blot and rescue experiments. In addition, xenograft tumor model was constructed to verify the phenotypes upon CAPRIN1 silencing.ResultsCaprin-1 expression was significantly elevated in both ESCA tumor tissues and cell lines compared with that in normal adjacent tissues and fibroblasts. Increased CAPRIN1 mRNA expression was significantly associated with clinical prognosis and diagnostic accuracy. The GO enrichment and KEGG pathway analysis CAPRIN1 might be related to immune-related terms, protein binding processes, and metabolic pathways. A significant positive correlation was observed between high Caprin-1 protein levels and lymph node metastasis (P = 0.031), ki-67 (P = 0.023), and 18F- FDG PET/CT parameters (SUVmax (P = 0.002) and SUV mean (P = 0.005)) in 55 ESCA patients. At cut-off values of SUVmax 17.71 and SUVmean 10.14, 18F- FDG PET/CT imaging predicted Caprin-1 expression in ESCA samples with 70.8% sensitivity and 77.4% specificity. In vitro and in vivo assays showed that Caprin-1 knockdown affected ESCA tumor growth. Silencing Caprin-1 inhibited ESCA cell proliferation and glycolysis, and decreased the expression of methyltransferase-like 3 (METTL3) and Wilms’ tumor 1-associating protein (WTAP). However, this effect could be partially reversed by the restoration of METTL3 and WTAP expression.ConclusionsOur data suggest that Caprin-1 could serve as a prognostic biomarker and has an oncogenic role in ESCA.

Metformin attenuates multiple myeloma cell proliferation and encourages apoptosis by suppressing METTL3-mediated m6A methylation of THRAP3, RBM25, and USP4

ABSTRACT Based on the results of epidemiological and preclinical studies, metformin can improve the prognosis of patients with malignant tumors. Studies have confirmed that metformin inhibits multiple myeloma (MM) cell proliferation and promotes apoptosis. Nevertheless, the specific mechanism remains to be elucidated. MM cells were intervened with different doses of metformin to detect cell proliferation and apoptosis. Western blotting and RT-qPCR were employed to assess the expression of METTL3, METTL14, WTAP, FTO, and ALKBH5 after metformin intervention. The microarray dataset GSE29023 was retrieved from the Gene Expression Omnibus (GEO) database and calculated using the R language (limma package) to authenticate differentially expressed genes (DEGs). The database for annotation, visualization, and integrated discovery (David) was applied for GO annotation analysis of DEGs. Subsequently, the string database and Cytoscape software were applied to construct protein–protein interaction (PPI) and DEM hub gene networks. Bioinformatics analysis and MeRIP were applied to predict and test METTL3-mediated m6A levels on mRNA of THRAP3, RBM25, and USP4 in METTL3 knocked-down cells. Then rescue experiments were performed to explore effects of METTL3 and THRAP3, RBM25, or USP4 on cell proliferation and apoptosis. The effect on MM cell xenograft tumor growth was observed by injection of metformin or/and overexpression of METTL3 in in vivo experiments. Metformin decreased cell proliferation and encouraged cell apoptosis in a dose-dependent manner. Global m6A modification was elevated in MM cells compared to normal cells, which was counteracted by metformin treatment. Furthermore, THRAP3, RBM25, and USP4 were identified as possible candidate genes for metformin treatment by GSE29023 data mining. METTL3 interference impaired m6A modification on mRNA of THRAP3, RBM25, and USP4 as well as expression levels. The mRNA stability and expression of THRAP3, RBM25, and USP4 was decreased after metformin treatment, which was reversed by METTL3 overexpression. THRAP3, RBM25 or USP4 knockdown reversed the assistance of METTL3 overexpression on the malignant behavior of MM cells. Finally, upregulation of METTL3 was shown to exert facilitative effects on xenograft tumor growth by blocking metformin injection. The present study demonstrates that metformin can repress the expression of THRAP3, RBM25, and USP4 by inhibiting METTL3-mediated m6A modification, which in turn hamper cell proliferation and promotes cell apoptosis. Abbreviations: multiple myeloma (MM), Gene Expression Omnibus (GEO), differentially expressed genes (DEGs), database for annotation, visualization and integrated discovery (David), protein–protein interaction (PPI), epithelial‑mesenchymal transition (EMT), methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), wilms tumor 1-associated protein (WTAP), methyltransferase like 16 (METTL16), acute myeloid leukemia (AML), non-small lung cancer (NSCLC), glioma stem cells (GSCs), normal bone marrow-derived plasma cells (nPCs), false discovery rate (FDR), biological process (BP), optical density (OD), horseradish peroxidase (HRP), M6A RNA immunoprecipitation assay (MeRIP).

WTAP-mediated m6A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis

An imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) is an important pathogenic mechanism underlying osteoporosis (OP). N6-methyladenosine (m6A) is the most common post-transcriptional modification in eukaryotic cells. The role of the Wilms’ tumor 1-associated protein (WTAP), a member of the m6A functional protein family, in regulating BMSCs differentiation remains unknown. We used patient-derived and mouse model-derived samples, qRT-PCR, western blot assays, ALP activity assay, ALP, and Alizarin Red staining to determine the changes in mRNA and protein levels of genes and proteins associated with BMSCs differentiation. Histological analysis and micro-CT were used to evaluate developmental changes in the bone. The results determined that WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. We used co-immunoprecipitation (co-IP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), RNA pulldown, and dual-luciferase assay to explore the direct mechanism. Mechanistically, the expression of WTAP increased during osteogenic differentiation and significantly promoted pri-miR-181a and pri-miR-181c methylation, which was recognized by YTHDC1, and increased the maturation to miR-181a and miR-181c. MiR-181a and miR-181c inhibited the mRNA expression of SFRP1, promoting the osteogenic differentiation of BMSCs. Our results demonstrated that the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axis regulated the differentiation fate of BMSCs, suggesting that it might be a potential therapeutic target for osteoporosis.

TEAD4 is a master regulator of high-risk nasopharyngeal carcinoma

The molecular basis underlying nasopharyngeal carcinoma (NPC) remains unclear. Recent progress in transcriptional regulatory network analysis helps identify the master regulator (MR) proteins that transcriptionally define malignant tumor phenotypes. Here, we investigated transcription factor-target interactions and identified TEA domain transcription factor 4 (TEAD4) as an MR of high-risk NPC. Precisely, TEAD4 promoted NPC migration, invasion and cisplatin resistance, depending on its autopalmitoylation. Mechanistically, YTHDF2 (YTH domain family 2) recognized WTAP (Wilms tumor 1-associating protein)-mediated TEAD4 m6A methylation to facilitate its stability and led to aberrant up-regulation of TEAD4. Up-regulated TEAD4 further drove NPC progression by transcriptionally activating BZW2 (basic leucine zipper and W2 domains 2) to induce the oncogenic AKT pathway. Moreover, the transcriptional activity of TEAD4 was independent of its canonical coactivators YAP/TAZ. Clinically, TEAD4 serves as an independent predictor of unfavorable prognosis and cisplatin response in NPC. Our data revealed the crucial role of TEAD4 in driving tumor malignancy, thus, may provide therapeutic vulnerability in NPC.

High expression of WTAP is related to poor prognosis in nasopharyngeal carcinoma.

Wilms' tumor 1-associated protein (WTAP), a component of the m6A methyltransferase complex, recruits the m6A methyltransferases METTL3 and METTL14 to the corresponding mRNA targets to participate in the formation of N6-methyladenosine. However, the molecular mechanism of WTAP in the tumorigenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. This study aimed to explore the prognostic value and biological function of WTAP in NPC. We assessed WTAP expression and its prognostic significance using microarray datasets from the Gene Expression Omnibus (GSE12452) database and 100 NPC tissues via bioinformatics analysis and immunohistochemistry (IHC), respectively. Moreover, gene ontology (GO) and gene set enrichment analysis (GSEA) were performed. In addition, the correlation of WTAP expression with the expression of immune cell biomarkers was analyzed. The results showed that WTAP expression was significantly overexpressed in NPC tissues in GSE12452. The overexpression of WTAP was validated by the external datasets including NPC tissues (GSE150430) and NPC cell lines (GSE39826). GO analysis suggested enrichment in the nucleoplasm (cellular component) and cell cycle (biological process). The GSEA revealed that differentially expressed genes were enriched in E2F-targets, Myc_targets_v1, G2M checkpoint, Myc_targets_v2, and Interferon-alpha-response. In IHC analysis, WTAP was upregulated in NPC tissues, and high levels of WTAP expression were significantly correlated with the advanced T stage (p=0.047) and advanced N stage (p=0.018). Cox regression demonstrated that WTAP overexpression was an independent biomarker of poor prognosis for overall survival (hazard ratio [HR], 4.747; 95% confidence interval [CI], 1.671-13.482; p=0.003). In IHC analysis, the expression of WTAP was positively correlated with CD206 (biomarker for M2 macrophages) (p=0.018) but negatively correlated with CD8a (biomarker for cytotoxic T cells) (p=0.001). In conclusion, WTAP is a promising prognostic biomarker and may participate in the regulation of immune cell infiltration in NPC.

N6-methyladenosine-related genes contribute to malignant progression, have clinical prognostic and neoadjuvant treatments response impact for breast cancer

N6-methyladenosine (m6A) methylation dysregulation contributes to tumorigenesis and breast cancer development. This study intends to conduct a comprehensive analysis for determining the clinical significance of m6A-related genes and establishing m6A-related gene-based risk signature to predict the clinical outcomes and neoadjuvant treatments response for breast cancer patients. The m6Avar database was utilized for downloading the m6A-regulated genes. The Cancer Genome Atlas (TCGA) database was utilized for downloading breast cancer patients’ RNA-Seq data and clinicopathological information. For determining the differentially expressed m6A-related gene, a one-way analysis of variance (ANOVA) was conducted. The interaction and correlation of m6A-related genes were evaluated using search tool for the retrieval of interacting genes/proteins (STRING) and Spearmen test. For determining clusters of breast cancer patients with different clinical outcomes, a consensus clustering analysis was conducted. We screened differentially expressed genes and functional enrichment pathways between subgroups utilizing gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). We constructed and verified a prognostic signature utilizing Cox regression analysis as well as a least absolute shrinkage and selection operator (LASSO) regression model. 286 genes were detected as significantly differentially expressed in different stages, including 3 m6A RNA methylation regulators, Wilms tumor 1-associating protein (WTAP), YT521-B homology (YTH) domain containing 2 (YTHDC2), and YTH domain family 2 (YTHDF2). A 13-gene prognostic signature was constructed and could predict the overall survival and the neoadjuvant treatments response in breast cancer patients. We categorized breast cancer patients into four groups based on m6A-associated RNAs expression. Significant differences were found in the overall survivals among the four clusters of patients. The biological processes and the key signaling pathways closely related to breast cancer have a close connection to the four clusters. This study confirmed that the m6A-related genes expression levels were highly associated with prognosis and neoadjuvant treatment response in breast cancer and constructed an effective m6A-related gene-based risk signature for predicting the prognosis of patients with breast cancer.

Prognostic and therapeutic implication of m6A methylation in Crohn disease

N6-methyladenosine (m6A) methylation has been reported to participate in inflammatory bowel disease (including Crohn disease [CD]). However, the prognostic and therapeutic implication of m6A methylation modification in CD is still unclear. Genomic information of CD patients was integrated to assess disease-related m6A regulators, and difference and correlation analyses of m6A regulators were explored by using the R packages. Next, CD patients were classified by the expression of differential and intersecting genes in m6A regulators, and difference and correlation analyses were conducted among immune infiltration and therapeutic responses. Finally, colon tissue resected from patients with CD were assessed to verify expression of Wilms tumor 1-associated protein (WTAP) and METTL14 from these m6A regulators. We identified 23 m6A regulators in CD patients. Difference analysis of these regulators showed that expression of METTL14, WTAP, RBM15 and YTHDF2/3 was upregulated in the treatment group compared with the control group, with expression of METTL3, YTHDF1, leucine-rich pentatricopeptide repeat motif-containing protein, HNRNPA2B1, IGF2BP1 and fat mass and obesity-associated protein downregulated. Moreover, RBM15, WTAP, leucine-rich pentatricopeptide repeat motif-containing protein, YTHDF1 and YTHDF3 were considered the characteristic genes of CD in m6A regulators. In addition, we identified 4 intersection genes of 3 m6A cluster patterns. Based on the expression of these intersection genes, difference analysis among m6A regulators indicated that the expression of 8 m6A regulators had statistical differences among the 3 geneCluster patterns. Assays of colon tissues from CD patients showed that expression of WTAP and METTL14 were higher in areas of stenosis than non-stenosis. m6A methylation modification might affect disease risk, immune infiltration and therapeutic responses in CD. Evaluating the expression of m6A regulators might provide insight into the prediction of disease prognosis and therapeutic responses.

WTAP dysregulation-mediated HMGN3-m6A modification inhibited trophoblast invasion in early-onset preeclampsia.

Early-onset preeclampsia (ePE) originates from abnormal implantation and placentation that involves trophoblast invasion, but its pathophysiology is not entirely understood. N6-methyladenosine (m6A) regulators mediate the progression of various cancers. The invasiveness of trophoblast cells is similar to that of tumor cells. However, little is known regarding the potential role of m6A modification in ePE and the underlying mechanism. This study aimed to explore the m6A level in placental tissue samples collected from ePE patients and to investigate whether m6A modification was an essential part of PE pathogenesis. The m6A level in placental tissue samples of 80 PE participants was examined. MeRIP-microarray, RNA-Seq, luciferase reporter assay, and RNA immunoprecipitation chip (RIP) assay were performed. The m6A level in the ePE group was significantly reduced compared with the control group. Wilms' tumor 1-associating protein (WTAP) regulated trophoblast cell migration and invasion. Mechanistically, the high mobility group nucleosomal binding domain 3 (HMGN3) gene was a target gene of WTAP in trophoblast (p < .05). WTAP enhanced the stability of HMGN3 mRNA through binding with its 3'-UTR m6A site(+485A, +522A). HMGN3 was recognized by m6A recognition protein insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which was inhibited when knocking down WTAP. Both m6A and WTAP levels were downregulated in ePE. The m6A modification mediated by WTAP/IGF2BP1/HMGN3 axis might contribute to abnormal trophoblast invasion. Our work provided a foundation for further exploration of RNA epigenetic regulatory patterns in ePE, and indicated a new treatment strategy for ePE.

N6-methyladenosine (m6A) RNA methylation mediated by methyltransferase complex subunit WTAP regulates amelogenesis

N6-methyladenosine (m6A) RNA methylation, one of the most widespread posttranscriptional modifications in eukaryotes, plays crucial roles in various developmental processes. The m6A modification process is catalyzed by a methyltransferase complex that includes Wilms tumor 1-associated protein (WTAP) as a key component. Whether the development of dental enamel is regulated by m6A RNA methylation in mammals remains unclear. Here, we reveal that WTAP is widely expressed from the early stage of tooth development. Specific inactivation of Wtap in mouse enamel epithelium by the Cre/loxp system leads to serious developmental defects in amelogenesis. In Wtap conditional KO mice, we determined that the differentiation of enamel epithelial cells into mature ameloblasts at the early stages of enamel development is affected. Mechanistically, loss of Wtap inhibits the expression of Sonic hedgehog (SHH), which plays an important role in the generation of ameloblasts from stem cells. Together, our findings provide new insights into the functional role of WTAP-mediated m6A methylation in amelogenesis in mammals.

Comprehensive Analysis Revealed the Potential Roles of N6-Methyladenosine (m6A) Mediating E. coli F18 Susceptibility in IPEC-J2 Cells.

Post-weaning diarrhea caused by enterotoxigenic Escherichia coli F18 (E. coli F18) causes significant economic losses for pig producers. N6-methyladenosine (m6A) is a highly abundant epitranscriptomic marker that has been found to be involved in regulating the resistance of host cells to pathogenic infection, but its potential role in E. coli F18-exposed intestinal porcine epithelial cells (IPEC-J2) remains undetermined. Here, we demonstrated that m6A and its regulators modulate E. coli F18 susceptibility. Briefly, we revealed that the Wilms' tumor 1-associating protein (WTAP) expressions were markedly elevated in IPEC-J2 cells upon E. coli F18 exposure. WTAP are required for the regulation of E. coli F18 adhesion in IPEC-J2 cells. Additionally, WTAP knockdown significantly suppressed m6A level at N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase (GCNT2) 3'UTR, resulting in the enhancement of TH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated GCNT2 mRNA stability. Subsequently, the altered GCNT2 expressions could inhibit the glycosphingolipid biosynthesis, thus improving resistance to E. coli F18 infection in IPEC-J2. Collectively, our analyses highlighted the mechanism behind the m6A-mediated management of E. coli F18 susceptibility, which will aid in the development of novel approaches that protect against bacterial diarrhea in piglets.

WTAP: a new player in postnatal BAT development

Abstract Understanding the mechanisms underlying brown fat development and metabolism can provide unique insights into the regulation of energy homeostasis. In a recent study published in Life Metabolism, Wang et al. established Wilms’ tumor 1-associating protein (WTAP), a key component in m 6A methyltransferase complex, as a new and essential regulator in the postnatal development and maturation of interscapular brown adipose tissue (iBAT).

The N6-methyladenosine writer WTAP contributes to the induction of immune tolerance post kidney transplantation by targeting regulatory T cells

AbstractN6-methyladenosine (m6A) modification is involved in diverse immunoregulation, while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP), an m6A writer, was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro, WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward naïve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region, leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression, while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice, as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function.

WTAP Targets the METTL3 m6A-Methyltransferase Complex to Cytoplasmic Hepatitis C Virus RNA to Regulate Infection.

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral life cycle. This life cycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms' tumor 1-associating protein (WTAP). WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production was independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV life cycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

WTAP regulates postnatal development of brown adipose tissue by stabilizing METTL3 in mice

Abstract Brown adipocyte maturation during postnatal development is essential for brown adipose tissue (BAT) to protect animals against cold. Impaired maturation of brown adipocytes leads to cold intolerance. However, the molecular mechanisms that determine the maturation of brown adipocytes during postnatal development are not fully understood. Here, we identify Wilms’ tumor 1-associating protein (WTAP) as an essential regulator in the postnatal development and maturation of BAT. BAT-specific knockout of Wtap (Wtap-BKO) severely impairs maturation of BAT in vivo by decreasing the expression of BAT-selective genes, leading to the whitening of interscapular BAT (iBAT). Single nucleus RNA-sequencing analysis shows the dynamic changes of cell heterogeneity in iBAT of Wtap-BKO mice. Adult mice with WTAP deficiency in BAT display hypothermic and succumb to acute cold challenge. Mechanistically, WTAP deficiency decreases m6A mRNA modification by reducing the protein stability of METTL3. BAT-specific overexpression of Mettl3 partially rescues the phenotypes observed in Wtap-BKO mice. These data demonstrate that WTAP/METTL3 plays an essential role in iBAT postnatal development and thermogenesis.

WTAP mediates the anti-inflammatory effect of Astragalus mongholicus polysaccharide on THP-1 macrophages.

Background: Astragalus mongholicus polysaccharides (APS) have anti-inflammatory, antioxidant and immunomodulatory effects. Recent studies have demonstrated the epigenetic regulation of N6-methyladenosine (m6A) in the development of inflammation. However, the effect of APS on m6A modification is unclear. Here, for the first time, we investigate the mechanism of m6A modification in APS regulation of THP-1 macrophage inflammation. Methods: We treated LPS-induced THP-1 macrophages with APS at different concentrations and times, and detected IL-6 mRNA and protein levels by quantitative real-time PCR (qRT-PCR) and western blot, respectively. The m6A modification level was detected by m6A quantification kit. The proteins that regulate m6A modification were screened by western blot. Wilms’ tumor 1-associating protein (WTAP) was overexpressed in APS-treated THP-1 macrophages and the m6A modification level and IL-6 expressions were detected. Results: These findings confirmed that APS significantly abolished LPS-induced IL-6 levels in THP-1 macrophages. Meanwhile, APS reduced m6A modification levels and WTAP gene expression in THP-1 macrophages. Further overexpression of WTAP can significantly reverse APS-induced m6A modification level and IL-6 expression. Mechanistically, APS regulates IL-6 expression through WTAP-mediated p65 nuclear translocation. Conclusion: Overall, our study suggested that WTAP mediates the anti-inflammatory effect of APS by regulating m6A modification levels in THP-1 macrophages. This study reveals a new dimension of APS regulation of inflammation at the epigenetic level.

Comprehensive analysis about prognostic and immunological role of WTAP in pan-cancer.

Background: Wilms tumor 1-associated protein (WTAP) plays a critical role in ribonucleic acid (RNA) methylation of N6 adenosine (m6A) modification, which is closely related with varieties of biological process. However, the role of WTAP in cancers remains to be determined. This study is designed to demonstrate the prognostic landscape of WTAP in pan-cancer and explore the relationship between WTAP expression and immune infiltration. Methods: Here, we investigated the expression level and prognostic role of WTAP in pan-cancer using multiple databases, including PrognoScan, GEPIA, and Kaplan-Meier Plotter. Then, applying the GEPIA and TIMER databases, we illustrated the correlations between WTAP expression and immune infiltration in tumors, especially liver hepatocellular carcinoma (LIHC), and esophageal carcinoma (ESCA). Results: WTAP had significant higher expression levels in tumor tissues of ESCA, LIHC, etc., while lower expression levels in those of bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), etc. And WTAP demonstrated multifaceted prognostic value in cancers. Of our interests, WTAP exerted a harmful effect on LIHC patient for overall survival (OS) and progression free survival (PFS). WTAP expression also significantly associated with the infiltration levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (DC) in LIHC but not ESCA. Furthermore, combined analysis about WTAP expression level and immune cell specific gene markers implied WTAP correlates with regulatory cells (T reg) infiltration in LIHC and ESCA. Conclusion: The m6A regulator WTAP can serve as a prognostic biomarker for certain tumor types in pan-cancer and potentially result from immune cell infiltration.