m6A RNA Modification Research Articles

N6-methyladenosine (m6A) RNA modification in the pathophysiology of heart failure: a narrative review.

Heart failure is the end-stage of various cardiovascular diseases. Recent progress in molecular biology has facilitated the understanding of the mechanisms of heart failure development at the molecular level. N6-adenosine methylation (m6A) is a post-transcriptional modification of RNA. Recent research work reported that m6A regulates gene expression and subsequently affects the activation of cell signaling pathways related to heart failure. Moreover, m6A regulators like methyltransferase-like 3 (METTL3) were reported to participate in myocardium hypertrophy. However, the current research work related to the role of m6A participating in the occurrence of heart failure is rare in some aspects like immune cell infiltration and diabetic heart diseases. Thus, it is reasonable to review the current achievements and provide further study orientation. We searched related literature using the keywords: m6A AND heart failure in PubMed, Web of Science and Medline. The language was confined to English. The published year of searched literature ranged from 2012 to 2022. The searched results were put into Endnote software for management. Two authors investigated the searching terms and reviewed the full text of selected terms. m6A and its regulators are involved in the metabolism of various types of RNAs. m6A modification can regulate various types of cell signaling pathways related to the heart failure via interaction with m6A regulators. m6A and its regulators broadly participate in the myocardium fibrosis, myocardium hypertrophy, myocardial cell apoptosis, and ischemic reperfusion injury. Specifically, m6A participates in the cell apoptosis via regulation of autophagy flux. However, the current research work does not have enough evidence to prove that m6A regulator played its specific effect on the target transcript via regulating the m6A level. m6A and its regulators participates in the progression of heart failure via modifying the RNA level. Future investigation of m6A should focus on the interaction between the m6A regulators and targeted transcript. Besides, the regulation role of m6A in immune cell infiltration and diabetic heart diseases should also be focused.

Chromatin-associated YTHDC1 coordinates heat-induced reprogramming of gene expression

Heat stress (HS) induces a cellular response leading to profound changes in gene expression. Here, we show that human YTHDC1, a reader of N6-methyladenosine (m6A) RNA modification, mostly associates to the chromatin fraction and that HS induces a redistribution of YTHDC1 across the genome, including to heat-induced heat shock protein (HSP) genes. YTHDC1 binding to m6A-modified HSP transcripts co-transcriptionally promotes expression of HSPs. In parallel, hundreds of the genes enriched in YTHDC1 during HS have their transcripts undergoing YTHDC1- and m6A-dependent intron retention. Later, YTHDC1 concentrates within nuclear stress bodies (nSBs) where it binds to m6A-modified SATIII non-coding RNAs, produced in an HSF1-dependent manner upon HS. These findings reveal that YTHDC1 plays a central role in a chromatin-associated m6A-based reprogramming of gene expression during HS. Furthermore, they support the model where the subsequent and temporary sequestration of YTHDC1 within nSBs calibrates the timing of this YTHDC1-dependent gene expression reprogramming.

IGF2BP3 is an essential N6-methyladenosine biotarget for suppressing ferroptosis in lung adenocarcinoma cells.

A lack of promising targets leads to poor prognosis in patients with lung adenocarcinoma (LUAD). Therefore, it is urgent to identify novel therapeutic targets. The importance of the N6-methyladenosine (m6A) RNA modification has been demonstrated in various types of tumors; however, knowledge of m6A-related proteins in LUAD is still limited. Here, we found that insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), an m6A reader protein, is highly expressed in LUAD and associated with poor prognosis. IGF2BP3 desensitizes ferroptosis (a new form of regulated cell death) in a manner dependent on its m6A reading domain and binding capacity to m6A-methylated mRNAs encoding anti-ferroptotic factors, including but not limited to glutathione peroxidase 4 (GPX4), solute carrier family 3 member 2 (SLC3A2), acyl-CoA synthetase long chain family member 3 (ACSL3), and ferritin heavy chain 1 (FTH1). After IGF2BP3 overexpression, expression levels and mRNA stabilities of these anti-ferroptotic factors were successfully sustained. Notably, significant correlations between SLC3A2, ACSL3, and IGF2BP3 were revealed in clinical LUAD specimens, further establishing the essential role of IGF2BP3 in desensitizing ferroptosis. Inducing ferroptosis has been gradually accepted as an alternative strategy to treat tumors. Thus, IGF2BP3 could be a potential target for the future development of new biomaterial-associated therapeutic anti-tumor drugs.

RNA m6A modification governs early human cardiomyocyte commitment

RNA m6A modification governs early human cardiomyocyte commitment

N6-methyladenosine-modified circular RNA QSOX1 promotes colorectal cancer resistance to anti-CTLA-4 therapy through induction of intratumoral regulatory T cells.

Colorectal cancer (CRC) is the 3rd most common cancer worldwide. CircRNAs are promising novel biomarkers for CRC. T regulatory (Treg) cells express the immune checkpoint receptor of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and promote tumor immunological tolerance. We therefore investigate the biological functions and mechanisms of circQSOX1 in CRC tumorigenesis; involvement of circQSOX1 in promoting Treg cell-mediated CRC immune escape in anti-CTLA-4 therapy. Bioinformatics analyses were performed for circQSOX1expressions, specific binding sites, and N6-methyladenosine (m6A) motifs of circQSOX1, thatwere further validated with a series of experiments. Functions of circQSOX1 in promoting CRC development, Treg cells-based immune escape, and anti-CTLA-4 therapy response were investigated both in vitro and in vivo. High circQSOX1 expression was associated with carcinogenesis and poor clinical outcome of CRC patients. METTL3-mediated RNA m6A modification on circQSOX1 could be read by IGF2BP2 in CRC cells. CircQSOX1 promoted CRC development by regulating miR-326/miR-330-5p/PGAM1 axis. CircQSOX1 regulated glycolysis and promoted immune escape of CRC cells, and inhibits anti-CTLA-4 therapy response in CRC patients. m6A-modified circQSOX1 facilitated CRC tumorigenesis by sponging miR-326 and miR-330-5p to promotes PGAM1 expression, which further promoted CRC immune escape by activating glycolysis and inactivating the anti-CTLA-4 therapy response of CRC. Combined treatment with sh-circQSOX1 and anti-CTLA-4 could be a strategy to overcome Treg cell-mediated CRC immune therapy resistance.

Shikonin targets to m6A-modified oxidative damage pathway to alleviate benzene-induced testicular injury.

Benzene exposure causes reproductive toxicity through oxidative damage. However, the specific mechanisms of benzene-induced testicular damage and the potential therapeutic drugs remain poorly understood. In the present study, C57BL/6J mice have been exposed to 0 and 150mg/kg benzene for four weeks. Then, we found that benzene exposure induced testicular damage in mice, mainly manifested by decreased testicular coefficients, abnormal semen parameters and HE-stained intraepithelial vacuolation. On the mechanism, benzene exposure activated Kelch Like ECH Associated Protein 1/Nuclear factor-erythroid 2 related factor 2 (Keap1/Nrf2) and NF-κB signaling pathway, then promoted apoptosis and inflammatory responses in testes. In vitro, massive reactive oxygen species (ROS) production and a large number of apoptotic cells were observed after 1,4-BQ treatment of GC-2cells. Furthermore, benzene altered the expression of three important RNA methylation modulator genes, methyltransferase-like 3 (Mettl3), AlkB homolog 5 (Alkbh5) and YTH domain containing 2 (Ythdc2). Moreover, both m6A modification and mRNA levels of NF-κB increased with benzene exposure. Inspiringly, shikonin alleviated benzene-induced male reproductive damage by targeting m6A-modified NF-κB in mice testes. Our study provides new insights into molecular mechanisms of RNA m6A modification in benzene-induced reproductive injury and directions to find potential drugs for the treatment of male infertility.

Long Noncoding RNA and mRNA m6A Modification Analyses of Periodontal Ligament Stem Cells from the Periodontitis Microenvironment Exposed to Static Mechanical Strain.

Periodontal ligament stem cells (PDLSCs) play important roles in orthodontic tooth movement (OTM) and can respond to mechanical stress. Our previous study demonstrated that periodontal ligament stem cells derived from periodontitis tissue (pPDLSCs) are more sensitive to static mechanical strain (SMS) than those derived from healthy tissue (hPDLSCs) and reported the long noncoding RNA (lncRNA) expression profiles of pPDLSCs exposed to SMS. An increasing number of lncRNAs have been reported by various studies to be associated with the osteogenic differentiation of mesenchymal stem cells. Many studies have demonstrated that the n6-methyladenosine (m6A) modification exerts important effects on lncRNA and mRNA regulation of cell behaviors. However, the regulatory effects of lncRNA and mRNA m6A modification on PDLSCs have not been studied. Therefore, we performed an m6A microarray assay with pPLDSCs and hPDLSCs exposed to 12% SMS and found that 143 lncRNAs and 739 mRNAs were differentially methylated. These RNAs were thought to be involved in multiple differentiation and inflammatory responses. Moreover, we found that METTL3, an essential protein in the m6A system, was expressed at lower levels in the strain-exposed pPDLSCs than in strain-exposed hPLDSCs, and METTL3 promoted the osteogenic differentiation of pPDLSCs.

RNA m6A methyltransferase METTL14 promotes the procession of non-small cell lung cancer by targeted CSF1R.

Non-small cell lung cancer (NSCLC) is one of the most malignant cancer types, characterized by a poor prognosis. N6-methyladenosine (m6A) is a prevalent internal modification of mRNA. METTL14, an RNA methyltransferase that mediates m6A modification, is implicated in mRNA biogenesis. However, the biomechanism of METTL14 in NSCLC is not very clear. Here, immunohistochemical (IHC) assay was employed to detect METTL14 in NSCLC tissues. The biological functions of METTL14 were demonstrated using cell transfection, cell proliferation assay, cell clone formation assay, cell cycle analysis, cell death analysis, transwell and wound healing assays. Transcriptome and methylated RNA immunoprecipitation (MERIP)-sequencing were used to explore the pathways and potential mechanism of METTL14 in NSCLC. RNA sequencing, METTL14 rip-sequencing, and METTL14 merip-sequencing were conducted to identify the potential targets of METTL14. METTL14 was significantly correlated with clinical pathological parameters of differentiation and M stage. Additionally, METTL14 promotes cell proliferation, induces cell death, and enhances cell migration and invasion in vitro. Transcriptome and MeRIP-sequencing reveal oncogenic mechanism of METTL14. RIP-sequencing highlights CSF1R and AKR1C1 as targets of METTL14. After validation with TCGA dataset, colony stimulating factor 1 receptor (CSF1R) showed significant positive coefficient with METTL14, and was presumed to be one target of METTl14 in lung cancer and verified by the cellular experiments. In conclusion, our results revealed the clinical significance of m6A RNA modification atlas, the function, and molecular targets CSF1R of METTL14 in NSCLC cell lines. The RNA m6A methyltransferase METTL14 promotes the progression of NSCLC by targeted CSF1R.

A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.

N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.

N6-methyladenosine (m6A) reader Pho92 is recruited co-transcriptionally and couples translation to mRNA decay to promote meiotic fitness in yeast.

N6- methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here, we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3'end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

The potential role of RNA N6-methyladenosine in primary Sjögren's syndrome.

The pathogenesis of primary Sjögren's syndrome (pSS) remains incompletely understood. The N6-methyladenosine (m6A) RNA modification, the most abundant internal transcript modification, has close associations with multiple diseases. This study aimed to investigate the role of m6A in patients with pSS. This study enrolled 44 patients with pSS, 50 age- and gender-matched healthy controls (HCs), and 11 age- and gender-matched patients with non-SS sicca. We detected the messenger RNA (mRNA) levels of m6A elements (including METTL3, WTAP, RBM15, ALKBH5, FTO, YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2), ISG15, and USP18 in peripheral blood mononuclear cells (PBMCs) from patients with pSS, patients with non-SS sicca, and HCs. The clinical characteristics and laboratory findings of patients with pSS and patients with non-SS sicca were also collected. We used binary logistic regression to determine if m6A elements were risk factors for pSS. The mRNA levels of m6A writers (METTL3 and RBM15), erasers (ALKBH5 and FTO), and readers (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2) were all significantly higher in PBMCs from patients with pSS than in HCs. The mRNA levels of m6A writers (METTL3 and WTAP) and readers (YTHDF2, YTHDF3, and YTHDC2) were lower in PBMCs from patients with pSS compared to patients with non-SS sicca. The expression of METTL3, RBM15, FTO, YTHDF1, YTHDF2, YTHDC1, and YTHDC2 was positively correlated with the level of C-reactive protein (CRP) of patients with pSS. The mRNA level of YTHDF1 in PBMCs from patients with pSS was negatively correlated with the EULAR Sjögren's syndrome disease activity index (ESSDAI) score. In patients with pSS, FTO, YTHDC1, and YTHDC2 were also related to white blood cells (WBCs), neutrophils, lymphocytes, and monocytes. Increased mRNA level of ALKBH5 in PBMCs was a risk factor for pSS, as determined by binary logistic regression analysis. The mRNA level of ISG15 was positively correlated with that of FTO, YTHDF2, YTHDF3, and YTHDC2 in patients with pSS. Compared with HCs, the expression of METTL3, RBM15, ALKBH5, FTO, YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2 was considerably higher in PBMCs from patients with pSS. In comparison with patients with non-SS sicca, the expression of METTL3, WTAP, YTHDF2, YTHDF3, and YTHDC2 was reduced in PBMCs from patients with pSS. The m6A elements correlating with clinical variables may indicate the disease activity and inflammation status of pSS. Elevated expression of ALKBH5 was a risk factor for pSS. The dynamic process of m6A modification is active in pSS. m6A elements (FTO, YTHDF2, YTHDF3, or YTHDC2) might target ISG15, stimulate the expression of ISG15, and activate the type I IFN signaling pathway, playing an active role in initiating the autoimmunity in pSS.

Non-coding RNA and n6-methyladenosine modification play crucial roles in neuropathic pain.

After peripheral nerve injury, pain signals are transmitted from primary sensory neurons in the dorsal root ganglion (DRG) to the central nervous system. Epigenetic modification affects neuropathic pain through alterations in the gene expression in pain-related areas and glial cell activation. Recent studies have shown that non-coding RNA and n6-methyladenosine (m6A) methylation modification play pivotal regulatory roles in the occurrence and maintenance of neuropathic pain. Dysregulation of the RNA m6A level via dynamic changes in methyltransferase and demethylase after central or peripheral nerve injury commonly regulates pain-associated genes, contributing to the induction and maintenance of neuropathic pain. The dynamic process has significant implications for the development and maintenance of neuropathic pain. However, the underlying mechanisms by which non-coding RNA and m6A RNA modification regulate neuropathic pain are not well-characterized. This article elucidates the multiple mechanisms of non-coding RNA and m6A methylation in the context of neuropathic pain, and summarizes its potential functions as well as recent advances.

Comparative Genomics and Functional Studies of Putative m6A Methyltransferase (METTL) Genes in Cotton.

N6-methyladenosine (m6A) RNA modification plays important regulatory roles in plant development and adapting to the environment, which requires methyltransferases to achieve the methylation process. However, there has been no research regarding m6A RNA methyltransferases in cotton. Here, a systematic analysis of the m6A methyltransferase (METTL) gene family was performed on twelve cotton species, resulting in six METTLs identified in five allotetraploid cottons, respectively, and three to four METTLs in the seven diploid species. Phylogenetic analysis of protein-coding sequences revealed that METTL genes from cottons, Arabidopsis thaliana, and Homo sapiens could be classified into three clades (METTL3, METTL14, and METTL-like clades). Cis-element analysis predicated the possible functions of METTL genes in G. hirsutum. RNA-seq data revealed that GhMETTL14 (GH_A07G0817/GH_D07G0819) and GhMETTL3 (GH_A12G2586/GH_D12G2605) had high expressions in root, stem, leaf, torus, petal, stamen, pistil, and calycle tissues. GhMETTL14 also had the highest expression in 20 and 25 dpa fiber cells, implying a potential role at the cell wall thickening stage. Suppressing GhMETTL3 and GhMETTL14 by VIGS caused growth arrest and even death in G. hirsutum, along with decreased m6A abundance from the leaf tissues of VIGS plants. Overexpression of GhMETTL3 and GhMETTL14 produced distinct differentially expressed genes (DEGs) in A. thaliana, indicating their possible divergent functions after gene duplication. Overall, GhMETTLs play indispensable but divergent roles during the growth of cotton plants, which provides the basis for the systematic investigation of m6A in subsequent studies to improve the agronomic traits in cotton.

The role of RNA modification in the generation of acquired drug resistance in glioma

Glioma is the most common malignant tumor in the central nervous system. The clinical treatment strategy is mainly surgery combined with concurrent temozolomide chemotherapy, but patients can develop drug resistance during treatment, which severely limits its therapeutic efficacy. Epigenetic regulation at the RNA level is plastic and adaptable, and it can induce a variety of tumor responses to drugs. The regulators of RNA modification include methyltransferases, demethylases, and methylation binding proteins; these are also considered to play an important role in the development, prognosis, and therapeutic response of gliomas, which provides a basis for finding new targets of epigenetic drugs and resetting the sensitivity of tumor cells to temozolomide. This review discusses the relationship between the development of adaptive drug resistance and RNA modification in glioma and summarizes the progress of several major RNA modification strategies in this field, especially RNA m6A modification, m5C modification, and adenosine-to-inosine editing.

METTL14 is required for exercise-induced cardiac hypertrophy and protects against myocardial ischemia-reperfusion injury

RNA m6A modification is the most widely distributed RNA methylation and is closely related to various pathophysiological processes. Although the benefit of regular exercise on the heart has been well recognized, the role of RNA m6A in exercise training and exercise-induced physiological cardiac hypertrophy remains largely unknown. Here, we show that endurance exercise training leads to reduced cardiac mRNA m6A levels. METTL14 is downregulated by exercise, both at the level of RNA m6A and at the protein level. In vivo, wild-type METTL14 overexpression, but not MTase inactive mutant METTL14, blocks exercise-induced physiological cardiac hypertrophy. Cardiac-specific METTL14 knockdown attenuates acute ischemia-reperfusion injury as well as cardiac dysfunction in ischemia-reperfusion remodeling. Mechanistically, silencing METTL14 suppresses Phlpp2 mRNA m6A modifications and activates Akt-S473, in turn regulating cardiomyocyte growth and apoptosis. Our data indicates that METTL14 plays an important role in maintaining cardiac homeostasis. METTL14 downregulation represents a promising therapeutic strategy to attenuate cardiac remodeling.

Abstract 13941: FTO Demethylase Mediates Altered N 6 -methyladenosine (m 6 A)-RNA Modification in Tyrosine Kinase Inhibitor-Induced Cardiotoxicity

Background: N 6 -methyladenosine (m 6 A) methylation is dynamically regulated by writers and erasers, and ultimately affects RNA splicing, translation, and stability after by recognized readers, thereby affecting disease progression. Sunitinib (SUN) is a tyrosine kinase inhibitor (TKI) widely used to treat different types of solid and hematological tumors, but its cardiotoxicity limits its clinical application. FTO is an m 6 A demethylase has been shown to play an important role in various heart diseases. In this study, we deciphered the methylation modification of m 6 A mRNA in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) post sunitinib treatment and assessed whether FTO regulated pyroptosis-related m 6 A methylated transcripts in SUN-treated hiPSC-CMs. Methods and Results: The global m 6 A RNA-methylation level was increased in SUN-treated hiPSC-CMs and was parallel with a positively correlated cellular damage level (LDH leakage). Through a genome-wide analysis of m 6 A mRNA methylation by MeRIP-Seq and RNA-seq, we identified a total of 2614 peaks with significant changes. qRT-PCR, immunofluorescence, and Western blot revealed that FTO was the most evidently changed m 6 A modifying enzymes in SUN-treated hiPSC-CMs (downregulated fold change being 0.43). Interestingly, the loss of FTO in hiPSC-CMs exposed to SUN was positively associated with cellular damage level and pyroptosis- associated NLRP3 and ASC transcripts levels. To further test if TKI-induced increase in m 6 A-RNA modification is mediated through FTO loss, hiPSC-CMs, overexpressed with ectopic FTO, showed significant restoration of m 6 A methylation level and normalization of pyroptosis-associated transcripts (NLRP3 and ASC) after exposure to SUN. To evaluate the effect of FTO on the methylation of RNA transcripts of NLRP3 and ASC, MeRIP assay demonstrated that FTO tightly controlled the methylation levels of NLRP3 and ASC transcripts, and demethylation of these transcripts by FTO accelerated their degradation rate. Conclusion: This study revealed that TKI-induced loss of FTO may mediate the decreased m 6 A-RNA methylation and TKI-induced cardiotoxicity pathogenesis by dysregulating the methylation and expression of pyroptotic transcripts NLRP3 and ASC.

IGF2BP2 promotes the progression of ovarian endometriosis by regulating m6A-modified MEIS2 and GATA6.

m6A-RNA modification mediated by the N6-methyladenosine RNA methylation-related molecule methyltransferase-like 3 has been implicated in the progression of endometriosis. However, the functions of other m6A regulators, especially in ovarian endometriosis, remain unknown. Three datasets (GSE7305, GSE7307, and GSE37837) with diagnosed ovarian endometriosis were extracted from the Gene Expression Omnibus database. Using bioinformatics methods such as Weighted Gene Co-expression Network Analysis, Gene Ontology analysis, protein-protein interaction, and correlation, hub genes were identified. Using dot blot and N6-methyladenosine-IP-qPCR, the total and individual N6-methyladenosine gene levels were quantified. On clinical ovarian ectopic and eutopic endometrium tissues, N6-methyladenosine RNA methylation sequencing was performed. To authenticate protein localization and expression level, immunohistochemical staining and western blot were conducted, respectively. The database Connectivity Map was used to predict small molecules with potential therapeutic effects. In ovarian endometriosis, the N6-methyladenosine "reader" molecule IGF2BP2 and related target genes MEIS2 and GATA6 were highly expressed. IGF2BP2 promoted the proliferation, migration, and invasion of ectopic endometrial stromal cells by stabilizing the mRNA of MEIS2 and GATA6. Synergistically, METTL3 and IGF2BP2 increased the N6-methyladenosine methylation of MEIS2 and GATA6. We developed five molecules (Mercaptopurine, MK-886, CP-863187, Canadine, and Securinine) that could be used to treat ovarian endometriosis based on IGF2BP2. Our findings provided additional support for a systematized understanding of the role of N6-methyladenosine RNA methylation in endometriosis and confirmed for the first time the mechanism of IGF2BP2 in promoting ovarian endometriosis. This provides the molecular foundation for potential future therapies for ovarian endometriosis. The data used to support the findings of this study are available from the corresponding author upon request.

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.

PSIII-3 Late-Breaking: Diisobutyl Phthalate (DIBP) at Low Doses Alters Gene Expression of Epigenetic Regulators in Bovine Granulosa Cells

Abstract Phthalates are ubiquitous environmental contaminants and impact reproduction and epigenetic mechanisms. The use of diisobutyl phthalate (DIBP) has increased in the last decade. However, studies about DIBP's effects on female reproductive physiology are limited. Methylation of DNA and RNA are epigenetic processes that regulate gene expression, cell biology, and reproductive functions. How DIBP exposure regulates these epigenetic mechanisms in ovarian follicular cells remains to be unveiled. We hypothesize that DIBP at low doses alters the expression of RNA and DNA methylation regulators in bovine granulosa cells (GC). Bovine GC was isolated from small antral follicles (1-5 mm on surface diameter) from ovaries obtained at a slaughterhouse. Cells were cultured in vitro until reaching an average 70% confluence and treated with DIBP at levels detected in human blood (0 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL) combined with hormones that regulate ovarian folliculogenesis: FSH or FSH+IGF1. Cells were cultured for 24h for total RNA isolation (n = 5 pools) or for 48h for cell counting (n=5 pools). Relative mRNA abundance of RNA N6 methyladenine (m6A) methylation machinery (m6A writers: METTL3, METTL14; m6A erasers: ALKBH5, FTO), RNA 5-methylcytosine (m5C) methylation regulators (NSUN2, TRDMT1), and DNA methylation players (DNA methyltransferases: DNMT1, DNMT3A, DNMT3B; DNA demethylases: TET1, TET2, TET3) was determined via qPCR using 18S as housekeeping gene and expressed as 2-ΔΔCt. Cell proliferation was analyzed by cell counting using a MUSE cell analyzer. Data were analyzed via one-way ANOVA with GLM procedures of SAS for Windows. Normality was tested using Shapiro-Wilk test procedures and data considered not normal were transformed to log2 (Y + 0.6). Data sets that showed non-gaussian distribution after log-transformation was analyzed via the Kruskal-Wallis test using GraphPad Prism 9.2.0. Cell proliferation was not affected by DIBP treatments in combination with FSH or with FSH+IGF1. In GC treated with FSH in combination with DIBP, there was no significant effect on relative mRNA abundance of RNA or DNA methylation players. In combination with FSH+IGF1, GC treated with DIBP had lower (P < 0.05) METTL14 relative mRNA abundance at 1 ng/mL and 10 ng/mL than at 0 ng/mL and 100 ng/mL. TRDMT1 relative mRNA abundance was lower (P < 0.05) in the DIBP group at 10 ng/mL in comparison to groups 0 ng/mL and 1 ng/mL, but no 100 ng/mL. In addition, there was no significant effect on the relative mRNA abundance of DNA methylation regulators. In summary, GC treated with FSH+IGF1 and DIBP at low doses alters the gene expression of RNA methylation players in m6A and m5C RNA modifications in a non-monotonic dose-response, but does not alter mRNA abundance of DNA methylation regulators.

Gene signature of m6A RNA regulators in diagnosis, prognosis, treatment, and immune microenvironment for cervical cancer

Continuing studies imply that m6A RNA modification is involved in the development of cervical cancer (CC), but lack strong support on recurrence and diagnosis prediction. In this research, a comprehensive analysis of 33 m6A regulators was performed to fulfill them. Here, we performed diagnostic and prognosis models and identified key regulators, respectively. Then the CC patients were separated into two clusters in accordance with 33 regulators, and participants in the cluster 1 had a worse prognosis. Subsequently, the m6AScore was calculated to quantify the m6A modification pattern based on regulators and we found that patients in cluster 1 had higher m6AScore. Afterwards, immune microenvironment, cell infiltration, escape analyses and tumor burden mutation analyses were executed, and results showed that m6AScore was correlated with them, but to a limited extent. Interestingly, HLAs and immune checkpoint expression, and immunophenoscore in patients with high-m6AScores were significantly lower than those in the low-m6AScore group. These suggested the m6AScores might be used to predict the feasibility of immunotherapy in patients. Results provided a distinctive perspective on m6A modification and theoretical basis for CC diagnosis, prognosis, clinical treatment strategies, and potential mechanism exploration.