m6A Reader Proteins Research Articles

Resveratrol Enhances Antioxidant and Anti-Apoptotic Capacities in Chicken Primordial Germ Cells through m6A Methylation: A Preliminary Investigation.

Avian primordial germ cells (PGCs) are essential in avian transgenic research, germplasm conservation, and disease resistance breeding. However, cultured PGCs are prone to fragmentation and apoptosis, regulated at transcriptional and translational levels, with N6-methyladenosine (m6A) being the most common mRNA modification. Resveratrol (RSV) is known for its antioxidant and anti-apoptotic properties, but its effects on PGCs and the underlying mechanisms are not well understood. This study shows that RSV supplementation in cultured PGCs improves cell morphology, significantly enhances total antioxidant capacity (p < 0.01), reduces malondialdehyde levels (p < 0.05), increases anti-apoptotic BCL2 expression, and decreases Caspase-9 expression (p < 0.05). Additionally, RSV upregulates the expression of m6A reader proteins YTHDF1 and YTHDF3 (p < 0.05). m6A methylation sequencing revealed changes in mRNA m6A levels after RSV treatment, identifying 6245 methylation sites, with 1223 unique to the control group and 798 unique to the RSV group. Combined analysis of m6A peaks and mRNA expression identified 65 mRNAs with significantly altered methylation and expression levels. Sixteen candidate genes were selected, and four were randomly chosen for RT-qPCR validation, showing results consistent with the transcriptome data. Notably, FAM129A and SFRP1 are closely related to apoptosis, indicating potential research value. Overall, our study reveals the protective effects and potential mechanisms of RSV on chicken PGCs, providing new insight into its use as a supplement in reproductive stem cell culture.

Abstract 5742: Therapeutic efficacy of CA102N in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a challenging cancer to treat due to its aggressive nature and late-stage diagnosis. Considering the lack of effective therapies, there is an urgent need to develop new and effective therapeutics to improve the prognosis of patients. CA102N is a carrier-mediated novel anticancer product where hyaluronic acid is conjugated to Nimesulide-NH2. CA102N is currently under development for the treatment of solid tumors, including colorectal cancer. CA102N was safe and well tolerated in the first-in-human trial in patients with locally advanced or metastatic solid tumors. In the present study, the in vitro anticancer activity of CA102N was investigated in K-ras mutant PANC-1 pancreatic cancer cells. The antitumor efficacy was also tested in vivo in the PANC-1 mice model. Results demonstrated that CA102N inhibits cell growth, cell migration, angiogenesis, and cell proliferation in both K-ras mutant PANC-1 pancreatic cancer cells or tumors. Administration of CA102N in PANC-1 xenografts resulted in a dose-dependent and statistically significant decrease in tumor growth at 9.8 mg/kg and 14.7 mg/kg Nim equivalents, respectively, compared to the vehicle control group. A significant decrease (80%) in liver metastasis was also noted in the PANC-1 orthotopic mice model at 19.6 mg/kg Nim equivalents dose level. Mechanistically, CA102N was found to suppress the PI3k/Akt/mTOR and Raf/MEK/ERK signaling pathways, in addition to reducing the expressions of the N6-methyladenosine (m6A) reader and writer proteins, OCT3/4 transcription factor and RAS levels in vivo. CA102N also modified the tumor stroma and improved drug permeability in vivo. Based on this compelling data, CA102N shows promise as a potential treatment for pancreatic cancer. Combination therapies and regimens are currently being explored in in vivo models as a strategy for clinical development. Citation Format: Louis Lin, Shuling Tu, Eskouhie Tchaparian. Therapeutic efficacy of CA102N in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5742.

YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation

Prostate cancer is a leading cause of cancer-related mortality in males. Dysregulation of RNA Adenine N-6 methylation (m6A) contributes to cancer malignancy. m6A on mRNA may affect mRNA splicing, turnover, transportation and translation. m6A exerts these effects, at least partly, through dedicated m6A reader proteins, including YTHDF2. YTHDF2 is necessary for development while its dysregulation is seen in various cancers including prostate cancer. However, the mechanism underlying the dysregulation and function of YTHDF2 in cancer remains elusive. Here, we find that the deubiquitinase OTUB1 increases YTHDF2 protein stability by inhibiting its ubiquitination. With in vivo and in vitro ubiquitination assays, OTUB1 is shown to block ubiquitin transfer to YTHDF2 independent of its deubiquitinase activity. Furthermore, analysis of functional transcriptomic data and m6A-sequencing data identifies PRSS8 as a potential tumor suppressor gene. OTUB1 and YTHDF2 decrease mRNA and protein levels of PRSS8, which is a trypsin-like serine protease. Mechanistically, YTHDF2 binds PRSS8 mRNA and promotes its degradation in an m6A-dependent manner. Further functional study on cellular and mouse models reveals PRSS8 is a critical downstream effector of the OTUB1-YTHDF2 axis in prostate cancer. We find in prostate cancer cells, PRSS8 decreases nuclear β-catenin level through E-cadherin, which is independent of its protease activity. Collectively, our study uncovers a key regulator of YTHDF2 protein stability and establishes a functional OTUB1-YTHDF2-PRSS8 axis in prostate cancer.

Atomic Mutagenesis of N6-Methyladenosine Reveals Distinct Recognition Modes by Human m6A Reader and Eraser Proteins.

N6-methyladenosine (m6A) is an important modified nucleoside in cellular RNA associated with multiple cellular processes and is implicated in diseases. The enzymes associated with the dynamic installation and removal of m6A are heavily investigated targets for drug research, which requires detailed knowledge of the recognition modes of m6A by proteins. Here, we use atomic mutagenesis of m6A to systematically investigate the mechanisms of the two human m6A demethylase enzymes FTO and ALKBH5 and the binding modes of YTH reader proteins YTHDF2/DC1/DC2. Atomic mutagenesis refers to atom-specific changes that are introduced by chemical synthesis, such as the replacement of nitrogen by carbon atoms. Synthetic RNA oligonucleotides containing site-specifically incorporated 1-deaza-, 3-deaza-, and 7-deaza-m6A nucleosides were prepared by solid-phase synthesis and their RNA binding and demethylation by recombinant proteins were evaluated. We found distinct differences in substrate recognition and transformation and revealed structural preferences for the enzymatic activity. The deaza m6A analogues introduced in this work will be useful probes for other proteins in m6A research.

ECT12, an YTH-domain protein, is a potential mRNA m6A reader that affects abiotic stress responses by modulating mRNA stability in Arabidopsis

N6-methyladenosine (m6A), the most abundant modification found in eukaryotic mRNAs, is interpreted by m6A “readers,” thus playing a crucial role in regulating RNA metabolism. The YT521-B homology-domain (YTHD) proteins, also known as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT), are recognized as m6A reader proteins in plants and animals. Among the 13 potential YTHD family proteins in Arabidopsis thaliana, the functions of only a few members are known. In this study, we determined the function of ECT12 (YTH11) as a potential m6A reader that plays a crucial role in response to abiotic stresses. The loss-of-function ect12 mutants showed no noticeable developmental defects under normal conditions but displayed hypersensitivity to salt or dehydration stress. The salt- or dehydration-hypersensitive phenotypes were correlated with altered levels of several m6A-modified stress-responsive transcripts. Notably, the increased or decreased transcript levels were associated with each transcript's reduced or enhanced decay, respectively. Electrophoretic mobility shift and RNA-immunoprecipitation assays showed that ECT12 binds to m6A-modified RNAs both in vitro and in planta, suggesting its role as an m6A reader. Collectively, these results indicate that the potential m6A reader ECT12 regulates the stability of m6A-modified RNA transcripts, thereby facilitating the response of Arabidopsis to abiotic stresses.

The Significance of N6-Methyladenosine RNA Methylation in Regulating the Hepatitis B Virus Life Cycle.

N6-methyladenosine (m6A) RNA methylation has recently emerged as a significant co-transcriptional modification involved in regulating various RNA functions. It plays a vital function in numerous biological processes. Enzymes referred to as m6A methyltransferases, such as the methyltransferaselike (METTL) 3-METTL14-Wilms tumor 1 (WT1)-associated protein (WTAP) complex, are responsible for adding m6A modifications, while m6A demethylases, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5), can remove m6A methylation. The functions of m6A-methylated RNA are regulated through the recognition and interaction of m6A reader proteins. Recent research has shown that m6A methylation takes place at multiple sites within hepatitis B virus (HBV) RNAs, and the location of these modifications can differentially impact the HBV infection. The addition of m6A modifications to HBV RNA can influence its stability and translation, thereby affecting viral replication and pathogenesis. Furthermore, HBV infection can also alter the m6A modification pattern of host RNA, indicating the virus's ability to manipulate host cellular processes, including m6A modification. This manipulation aids in establishing chronic infection, promoting liver disease, and contributing to pathogenesis. A comprehensive understanding of the functional roles of m6A modification during HBV infection is crucial for developing innovative approaches to combat HBV-mediated liver disease. In this review, we explore the functions of m6A modification in HBV replication and its impact on the development of liver disease.

Dysregulation of RNA modification systems in clinical populations with neurocognitive disorders.

The study of modified RNA known as epitranscriptomics has become increasingly relevant in our understanding of disease-modifying mechanisms. Methylation of N6 adenosine (m6A) and C5 cytosine (m5C) bases occur on mRNAs, tRNA, mt-tRNA, and rRNA species as well as non-coding RNAs. With emerging knowledge of RNA binding proteins that act as writer, reader, and eraser effector proteins, comes a new understanding of physiological processes controlled by these systems. Such processes when spatiotemporally disrupted within cellular nanodomains in highly specialized tissues such as the brain, give rise to different forms of disease. In this review, we discuss accumulating evidence that changes in the m6A and m5C methylation systems contribute to neurocognitive disorders. Early studies first identified mutations within FMR1 to cause intellectual disability Fragile X syndromes several years before FMR1 was identified as an m6A RNA reader protein. Subsequently, familial mutations within the m6A writer gene METTL5, m5C writer genes NSUN2, NSUN3, NSUN5, and NSUN6, as well as THOC2 and THOC6 that form a protein complex with the m5C reader protein ALYREF, were recognized to cause intellectual development disorders. Similarly, differences in expression of the m5C writer and reader effector proteins, NSUN6, NSUN7, and ALYREF in brain tissue are indicated in individuals with Alzheimer's disease, individuals with a high neuropathological load or have suffered traumatic brain injury. Likewise, an abundance of m6A reader and anti-reader proteins are reported to change across brain regions in Lewy bodies diseases, Alzheimer's disease, and individuals with high cognitive reserve. m6A-modified RNAs are also reported significantly more abundant in dementia with Lewy bodies brain tissue but significantly reduced in Parkinson's disease tissue, whilst modified RNAs are misplaced within diseased cells, particularly where synapses are located. In parahippocampal brain tissue, m6A modification is enriched in transcripts associated with psychiatric disorders including conditions with clear cognitive deficits. These findings indicate a diverse set of molecular mechanisms are influenced by RNA methylation systems that can cause neuronal and synaptic dysfunction underlying neurocognitive disorders. Targeting these RNA modification systems brings new prospects for neural regenerative therapies.

The Proteins of mRNA Modification: Writers, Readers, and Erasers.

Over the past decade, mRNA modifications have emerged as important regulators of gene expression control in cells. Fueled in large part by the development of tools for detecting RNA modifications transcriptome wide, researchers have uncovered a diverse epitranscriptome that serves as an additional layer of gene regulation beyond simple RNA sequence. Here, we review the proteins that write, read, and erase these marks, with a particular focus on the most abundant internal modification, N 6-methyladenosine (m6A). We first describe the discovery of the key enzymes that deposit and remove m6A and other modifications and discuss how our understanding of these proteins has shaped our views of modification dynamics. We then review current models for the function of m6A reader proteins and how our knowledge of these proteins has evolved. Finally, we highlight important future directions for the field and discuss key questions that remain unanswered. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Ythdf2 regulates cardiac remodeling through its mRNA target transcripts

m6A mRNA methylation controls cardiomyocyte function and increased overall m6A levels are a stereotyping finding in heart failure independent of the underlying etiology. However, it is largely unknown how the information is read by m6A reader proteins in heart failure. Here we show that the m6A reader protein Ythdf2 controls cardiac function and identified a novel mechanism how reader proteins control gene expression and cardiac function. Deletion of Ythdf2 in cardiomyocytes in vivo leads to mild cardiac hypertrophy, reduced heart function, and increased fibrosis during pressure overload as well as during aging. Similarly, in vitro the knockdown of Ythdf2 results in cardiomyocyte growth and remodeling. Mechanistically, we identified the eucaryotic elongation factor 2 as post-transcriptionally regulated by Ythdf2 using cell type specific Ribo-seq data. Our study expands our understanding on the regulatory functions of m6A methylation in cardiomyocytes and how cardiac function is controlled by the m6A reader protein Ythdf2.

M6A reader proteins: the executive factors in modulating viral replication and host immune response.

N6-Methyladenosine (m6A) modification is the most abundant covalent modification of RNA. It is a reversible and dynamic process induced by various cellular stresses including viral infection. Many m6A methylations have been discovered, including on the genome of RNA viruses and on RNA transcripts of DNA viruses, and these methylations play a positive or negative role on the viral life cycle depending on the viral species. The m6A machinery, including the writer, eraser, and reader proteins, achieves its gene regulatory role by functioning in an orchestrated manner. Notably, data suggest that the biological effects of m6A on target mRNAs predominantly depend on the recognition and binding of different m6A readers. These readers include, but are not limited to, the YT521-B homology (YTH) domain family, heterogeneous nuclear ribonucleoproteins (HNRNPs), insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), and many others discovered recently. Indeed, m6A readers have been recognized not only as regulators of RNA metabolism but also as participants in a variety of biological processes, although some of these reported roles are still controversial. Here, we will summarize the recent advances in the discovery, classification, and functional characterization of m6A reader proteins, particularly focusing on their roles and mechanisms of action in RNA metabolism, gene expression, and viral replication. In addition, we also briefly discuss the m6A-associated host immune responses in viral infection.

M6A readers ECT2/ECT3/ECT4 enhance mRNA stability through direct recruitment of the poly(A) binding proteins in Arabidopsis

BackgroundRNA N6-methyladenosine (m6A) modification is critical for plant growth and crop yield. m6A reader proteins can recognize m6A modifications to facilitate the functions of m6A in gene regulation. ECT2, ECT3, and ECT4 are m6A readers that are known to redundantly regulate trichome branching and leaf growth, but their molecular functions remain unclear.ResultsHere, we show that ECT2, ECT3, and ECT4 directly interact with each other in the cytoplasm and perform genetically redundant functions in abscisic acid (ABA) response regulation during seed germination and post-germination growth. We reveal that ECT2/ECT3/ECT4 promote the stabilization of their targeted m6A-modified mRNAs, but have no function in alternative polyadenylation and translation. We find that ECT2 directly interacts with the poly(A) binding proteins, PAB2 and PAB4, and maintains the stabilization of m6A-modified mRNAs. Disruption of ECT2/ECT3/ECT4 destabilizes mRNAs of ABA signaling-related genes, thereby promoting the accumulation of ABI5 and leading to ABA hypersensitivity.ConclusionOur study reveals a unified functional model of m6A mediated by m6A readers in plants. In this model, ECT2/ECT3/ECT4 promote stabilization of their target mRNAs in the cytoplasm.

YBX2 modulates mRNA stability via interaction with YTHDF2 in endometrial cancer cells

RNA-binding proteins (RBPs) fine-tune gene expression by modulating RNA stability, translation, and degradation. RBPs are involved in the development of endometrial cancer. In particular, Y-box-binding protein 2 (YBX2), a germ cell-specific member of the YBX family, has been reported to maintain cancer stem cell-like phenotypes in endometrial cancer. However, the mechanism by which YBX2 modulates mRNA stability in endometrial cancer cells remains unknown. In this study, we examined the effects of the ectopic expression of YBX2 in endometrial adenocarcinoma-derived Ishikawa cells. We found that elevated levels of YBX2 delayed cell proliferation, without increasing cell apoptosis. Transcriptomic analysis revealed disturbances in gene expression caused by YBX2. Interestingly, heat shock protein family A (Hsp70) member 6 (HSPA6) levels were downregulated due to the reduced mRNA stability after YBX2 binding. YBX2 facilitated the formation of relatively stable cytoplasmic granules in tumor cells via its mRNA binding domain. Moreover, N6-methyladenosine (m6A) reader proteins are recruited by YBX2 granules via the cold-shock domain. Notably, knockdown of YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2), an m6A reader, ameliorated the reduction in HSPA6 mRNA levels induced by YBX2, indicating the synergistic effects of YBX2 and YTHDF2 on mRNA stability. Therefore, YBX2 regulates RNA stability by interacting with the m6A reader proteins.

YTHDF2 Is Downregulated in Response to Host Shutoff Induced by DNA Virus Infection and Regulates Interferon-Stimulated Gene Expression.

Recent studies have begun to reveal the complex and multifunctional roles of N6-methyladenosine (m6A) modifications and their associated writer, reader, and eraser proteins in infection by diverse RNA and DNA viruses. However, little is known about their regulation and functions during infection by several viruses, including poxviruses. Here, we show that members of the YTH Domain Family (YTHDF), in particular YTHDF2, are downregulated as the prototypical poxvirus, vaccinia virus (VacV) enters later stages of replication in a variety of natural target cell types, but not in commonly used transformed cell lines wherein the control of YTHDF2 expression appears to be dysregulated. YTHDF proteins also decreased at late stages of infection by herpes simplex virus 1 (HSV-1) but not human cytomegalovirus, suggesting that YTHDF2 is downregulated in response to infections that induce host shutoff. In line with this idea, YTHDF2 was potently downregulated upon infection with a VacV mutant expressing catalytically inactive forms of the decapping enzymes, D9 and D10, which fails to degrade dsRNA and induces a protein kinase R response that itself inhibits protein synthesis. Overexpression and RNAi-mediated depletion approaches further demonstrate that YTHDF2 does not directly affect VacV replication. Instead, experimental downregulation of YTHDF2 or the related family member, YTHDF1, induces a potent increase in interferon-stimulated gene expression and establishes an antiviral state that suppresses infection by either VacV or HSV-1. Combined, our data suggest that YTHDF2 is destabilized in response to infection-induced host shutoff and serves to augment host antiviral responses. IMPORTANCE There is increasing recognition of the importance of N6-methyladenosine (m6A) modifications to both viral and host mRNAs and the complex roles this modification plays in determining the fate of infection by diverse RNA and DNA viruses. However, in many instances, the functional contributions and importance of specific m6A writer, reader, and eraser proteins remains unknown. Here, we show that natural target cells but not transformed cell lines downregulate the YTH Domain Family (YTHDF) of m6A reader proteins, in particular YTHDF2, in response to shutoff of protein synthesis upon infection with the large DNA viruses, vaccinia virus (VacV), or herpes simplex virus type 1. We further reveal that YTHDF2 downregulation also occurs as part of the host protein kinase R response to a VacV shutoff mutant and that this downregulation of YTHDF family members functions to enhance interferon-stimulated gene expression to create an antiviral state.

Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export.

Skeletal muscle stem cells (also known as satellite cells [SCs]) are essential for muscle regeneration and the regenerative activities of SCs are intrinsically governed by gene regulatory mechanisms, but the post-transcriptional regulation in SCs remains largely unknown. N(6)-methyladenosine (m6A) modification of RNAs is the most pervasive and highly conserved RNA modification in eukaryotic cells; it exerts powerful impact on almost all aspects of mRNA processing that is mainly endowed by its binding with m6A reader proteins. In this study, we investigate the previously uncharacterized regulatory roles of YTHDC1, an m6A reader in mouse SCs. Our results demonstrate that YTHDC1 is an essential regulator of SC activation and proliferation upon acute injury-induced muscle regeneration. The induction of YTHDC1 is indispensable for SC activation and proliferation; thus, inducible YTHDC1 depletion almost abolishes SC regenerative capacity. Mechanistically, transcriptome-wide profiling using LACE-seq in both SCs and mouse C2C12 myoblasts identifies m6A-mediated binding targets of YTHDC1. Next, splicing analysis defines splicing mRNA targets of m6A-YTHDC1. Furthermore, nuclear export analysis also leads to the identification of potential mRNA export targets of m6A-YTHDC1 in SCs and C2C12 myoblasts;interestingly, some mRNAs can be regulated at both splicing and export levels. Lastly, we map YTHDC1 interacting protein partners in myoblasts and unveil a myriad of factors governing mRNA splicing, nuclear export, and transcription, among which hnRNPG appears to be a bona fide interacting partner of YTHDC1. Altogether, our findings uncover YTHDC1 as an essential factor controlling SC regenerative ability through multifaceted gene regulatory mechanisms in mouse myoblast cells.

Critical Roles of METTL3 in Translation Regulation of Cancer.

Aberrant translation, a characteristic feature of cancer, is regulated by the complex and sophisticated RNA binding proteins (RBPs) in the canonical translation machinery. N6-methyladenosine (m6A) modifications are the most abundant internal modifications in mRNAs mediated by methyltransferase-like 3 (METTL3). METTL3 is commonly aberrantly expressed in different tumors and affects the mRNA translation of many oncogenes or dysregulated tumor suppressor genes in a variety of ways. In this review, we discuss the critical roles of METTL3 in translation regulation and how METTL3 and m6A reader proteins in collaboration with RBPs within the canonical translation machinery promote aberrant translation in tumorigenesis, providing an overview of recent efforts aiming to 'translate' these results to the clinic.

Low expression of m6A reader YTHDC1 promotes progression of ovarian cancer via PIK3R1/STAT3/GANAB axis.

Background: N6-Methyladenosine (m6A) is considered to be the most prevalent and abundant internal modification observed in mRNA between viruses and mammals. As a reversible epigenetic modification, m6A controls gene expression in diverse physiological and pathological processes. Accumulating evidence in recent years reveals that aberrant expression of m6A reader proteins may have tumor-suppressing or carcinogenic functions. However, the biological role and mechanism of m6A reader YTH Domain Containing 1 (YTHDC1) in ovarian cancer progression remain inadequately understood. Methods: Quantitative RT-PCR, immunohistochemistry, Western blot, and bioinformatics analyses were undertaken for studying the YTHDC1 expression in ovarian cancer. In vitro and in vivo models were used to examine the role of YTHDC1. RNA sequencing, RNA immunoprecipitation sequencing, m6A-modified RNA immunoprecipitation, actinomycin-D assay, chromatin immunoprecipitation, and Western blot were used in the investigation the regulatory mechanisms among YTHDC1, Signal Transducer and Activator of Transcription 3 (STAT3), Phosphoinositide-3-Kinase Regulatory Subunit 1 (PIK3R1), and Glucosidase II Alpha Subunit (GANAB). Results: Here, we found that YTHDC1 expression is decreased in ovarian cancer. Overexpression of YTHDC1 inhibited ovarian cancer development both in vivo and in vitro. Mechanistically, PIK3R1 was identified to be the direct target for YTHDC1. YTHDC1 enhanced PIK3R1 stability in an m6A-dependent manner, which subsequently inhibited GANAB expression in the N-glycan biosynthesis via the STAT3 signaling. Conclusions: Our findings unveil YTHDC1 as a tumor suppressor in the progression of ovarian cancer and as a potential prognostic biomarker that could serve as a target in ovarian cancer treatment.

Potential impact of WTAP and YTHDF2 on tumor immunity in lung adenocarcinoma.

WTAP and N6-methyladenosine (m6A) reader proteins (YTHDF2) are N6-methyladenosine (m6A) methyltransferase and m6A reading proteins, respectively. In recent years, the tumor immune environment has received more and more attention in the progress and treatment of cancer. The aim of this study was to investigate the relationship between N6-methyladenosine (m6A) methyltransferase (WTAP)/YTHDF2 and the immunological characteristics of lung adenocarcinoma (LUAD). Based on the expression of WTAP and YTHDF2 in the cancer genome atlas (TCGA) and gene expression omnibus (GEO) database, LUAD patients were divided into 2 clusters by coherently clustering method, and performed gene set enrichment analysis (GSEA) to identify the functional differences. Immunoinvasion analysis was performed using ESTIMATE, CIBERSORT, and single-sample GSEA (ssGSEA), and expression of immune checkpoint inhibitors (ICIs) targets was assessed, while tumor mutation burden (TMB) was calculated in tumor samples. Weighted gene co-expression network analysis (WGCNA) was used to identify the genes related to both WTAP/YTHDF2 expression and immunity. The immunological characteristics between the 2 clusters were externally verified based on GSE39582. The expression of WTAP was higher in cluster 1 and YTHDF2 was lower, but it was opposite in cluster 2. Cluster 1 had stronger immune infiltration, more ICIs target expression, more TMB. In addition, WGCNA identified 22 genes associated with WTAP/YTHDF2 expression and immune score, including TIM3 (HAVCR2) and CD86. WTAP and YTHDF2 influence immune contexture and may be novel prognostic and druggable targets associated with the immune system of LUAD.

Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells.

RNA-binding proteins (RBPs) regulate nearly every aspect of mRNA processing and are important regulators of gene expression in cells. However, current methods for transcriptome-wide identification of RBP targets are limited, since they examine only a single RBP at a time and do not provide information on the individual RNA molecules that are bound by a given RBP. Here, we overcome these limitations by developing TRIBE-STAMP, an approach for single-molecule detection of the target RNAs of two RNA binding proteins simultaneously in cells. We applied TRIBE-STAMP to the cytoplasmic m6A reader proteins YTHDF1, YTHDF2, and YTHDF3 and discovered that individual mRNA molecules can be bound by more than one YTHDF protein throughout their lifetime, providing new insights into the function of YTHDF proteins in cells. TRIBE-STAMP is a highly versatile approach that enables single-molecule analysis of the targets of RBP pairs simultaneously in the same cells.

Downregulation of the m6A reader protein YTHDC1 leads to islet β-cell failure and diabetes

N6-methyladenosine (m6A) methyltransferase writer proteins (METTL3/METTL14) have been shown to regulate β-cell function and diabetes. However, whether and which m6A reader proteins regulate β-cell function and the pathogenesis of diabetes are largely unknown. In this study, we showed that YTHDC1 (YTH domain-containing protein 1), a key m6A nuclear reader protein, plays an essential role in maintaining β-cell function. YTHDC1 is downregulated in islet β cells in type 2 diabetes, which is due to lipotoxicity and chronic inflammation. β-Cell specific deletion of Ythdc1 results in β-cell failure and diabetes, which is likely due to the decreased expression of β-cell specific transcription factors and insulin secretion-related genes. Taken together, YTHDC1 is required for maintaining β-cell function, and the downregulation of YTHDC1 leads to β-cell failure and diabetes.

M6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-β-induced epithelial–mesenchymal transition of lung cancer cells

N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during transforming growth factor-beta-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and nonredundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by methyltransferase-like 3 (METTL3) m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during transforming growth factor-beta-induced EMT of lung cancer cells.