bims-rimeca 2021-07-25 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2021–07–25
35 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

The functions and prognostic values of m6A RNA methylation regulators in thyroid carcinoma.
N6-Methyladenosine RNA Demethylase FTO Promotes Gastric Cancer Metastasis by Down-Regulating the m6A Methylation of ITGB1.
IDH2 contributes to tumorigenesis and poor prognosis by regulating m6A RNA methylation in multiple myeloma.
Cigarette smoking induces aberrant N6-methyladenosine of DAPK2 to promote non-small cell lung cancer progression by activating NF-κB pathway.
N6-Methyladenosine RNA modification in cerebrospinal fluid as a novel potential diagnostic biomarker for progressive multiple sclerosis.
m6A Regulator-Mediated Methylation Modification Model Predicts Prognosis, Tumor Microenvironment Characterizations and Response to Immunotherapies of Clear Cell Renal Cell Carcinoma.
Comprehensive Analysis of the Immune Infiltrates and PD-L1 of m6A RNA Methylation Regulators in Hepatocellular Carcinoma.
m6A Regulator-Associated Modification Patterns and Immune Infiltration of the Tumor Microenvironment in Hepatocarcinoma.
Knockdown of METTL3 inhibits enterovirus 71 induced apoptosis of mouse Schwann cell through regulation of autophagy.
Crosstalk between epitranscriptomic and epigenetic mechanisms in gene regulation.
m6A RNA methylation regulates promoter- proximal pausing of RNA polymerase II.
Conditional deficiency of m6A methyltransferase Mettl14 in substantia nigra alters dopaminergic neuron function.
N6-methyladenosine (m6A)-mediated up-regulation of long noncoding RNA LINC01320 promotes the proliferation, migration, and invasion of gastric cancer via miR495-5p/RAB19 axis.
HNRNPA2B1 Affects the Prognosis of Esophageal Cancer by Regulating the miR-17-92 Cluster.
M6A Demethylase ALKBH5 Regulates PD-L1 Expression and Tumor Immunoenvironment in Intrahepatic Cholangiocarcinoma.
Identification of the m6A RNA Methylation Regulators WTAP as a Novel Prognostic Biomarker and Genomic Alterations in Cutaneous Melanoma.
The Emerging Clinical Application of m6A RNA Modification in Inflammatory Bowel Disease and Its Associated Colorectal Cancer.
Widespread remodeling of the m6A RNA-modification landscape by a viral regulator of RNA processing and export.
Transcriptional regulation of N6-methyladenosine orchestrates sex-dimorphic metabolic traits.
LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC.
m6A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection.
Hub Long Noncoding RNAs with m6A Modification for Signatures and Prognostic Values in Kidney Renal Clear Cell Carcinoma.
Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore.
The role m6A RNA methylation is CNS development and glioma pathogenesis.
Prognostic signature and immune efficacy of m1 A-, m5 C- and m6 A-related regulators in cutaneous melanoma.
MicroRNA-211 attenuates cell proliferation in T-cell lymphoblastic lymphoma through targeting TCF12.
RNA 5-methylcytosine modification and its emerging role as an epitranscriptomic mark.
miR-302a-3p suppresses melanoma cell progression via targeting METTL3.
Active genic machinery for epigenetic RNA modifications in bees.
RBM15-mediated N6-methyladenosine modification affects COVID-19 severity by regulating the expression of multitarget genes.
Epitranscriptomics: A New Layer of microRNA Regulation in Cancer.
Single-cell Imaging of m6A modified RNA Using m6A-specific In Situ Hybridization mediated Proximity Ligation Assay (m6AISH-PLA).
ModPhred: an integrative toolkit for the analysis and storage of nanopore sequencing DNA and RNA modification data.
ALKBH5 Promotes the Proliferation of Glioma Cells via Enhancing the mRNA Stability of G6PD.
Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus.

Cancer Cell Int. 2021 Jul 19. 21(1): 385

The functions and prognostic values of m6A RNA methylation regulators in thyroid carcinoma.

Zhi-Hao Yu, Shao-Ting Feng, Di Zhang, Xu-Chen Cao, Yue Yu, Xin Wang.

   BACKGROUND: N6-Methyladenosine (m6A) is the most common RNA modification and regulates RNA splicing, translation, translocation, and stability. Aberrant expression of m6A has been reported in various types of human cancers. m6A RNA modification is dynamically and reversibly mediated by different regulators, including methyltransferase, demethylases, and m6A binding proteins. However, the role of m6A RNA methylation regulators in thyroid cancer remains unknown. The aim of this study is to investigate the effect of the 13 main m6A RNA modification regulators in thyroid carcinoma.
METHODS: We obtained clinical data and RNA sequencing data of 13 m6A RNA methylation regulators from The Cancer Genome Atlas (TCGA) THCA database. We performed consensus clustering to identify the clinical relevance of m6A RNA methylation regulators in thyroid carcinoma. Then we used LASSO Cox regression analysis to generate a prognostic signature based on m6A RNA modification regulator expression. Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Gene Set Enrichment Analyses were performed to explore differential cellular processes and signaling pathways between the two groups based on risk signature.
RESULTS: We found that most of the m6A RNA modification regulators are down-regulated in 450 patients with thyroid carcinoma. We derived a three m6A RNA modification regulator genes-based risk signature (FTO, RBM15 and KIAA1429), that is an independent prognostic biomarker in patients with thyroid carcinoma. Moreover, we found that this risk signature could better predict outcome in male than female. Functional research in vitro demonstrated that the m6A RNA methylation regulators involved in the model acted significant role in the proliferation and migration of thyroid cancer cells.
CONCLUSIONS: Our study revealed the influence of m6A RNA methylation regulators on thyroid carcinoma through biological experiments and three-gene prognostic model.

Keywords:  Experimental validation; Gene signature; Prognosis; TCGA; Thyroid carcinoma; m6A RNA methylation regulator

DOI:  https://doi.org/10.1186/s12935-021-02090-9
Front Oncol. 2021 ;11 681280

N6-Methyladenosine RNA Demethylase FTO Promotes Gastric Cancer Metastasis by Down-Regulating the m6A Methylation of ITGB1.

Duo Wang, Xiujuan Qu, Wenqing Lu, Yizhe Wang, Yue Jin, Kezuo Hou, Bowen Yang, Ce Li, Jianfei Qi, Jiawen Xiao, Xiaofang Che, Yunpeng Liu.

  Abnormal RNA m6A methylation is known to lead to the occurrence and progression of multiple cancers including gastric cancer (GC). However, the integrative effects of all m6A methylation regulators on GC prognosis are unclear. Our research aimed to globally analyze the prognosis values of all 33 m6A RNA methylation regulators in GC by univariate and multivariate Cox regression analyses. Among all 33 m6A RNA methylation regulators, fat mass and obesity-associated protein (FTO), an m6A demethylase, was identified as a key prognostic risk factor on overall survival (OS) of GC patients. It was found that FTO could promote GC cell migration and invasion abilities, and we predicted that ITGB1 was a demethylated target of FTO. Knockdown (KD) of FTO significantly down-regulated ITGB1 expression at both mRNA and protein levels and augmented ITGB1 mRNA m6A modification level. Moreover, overexpression (OE) of ITGB1 could partially reverse FTO-KD-inhibited migration and invasion of GC cells. Our study found that FTO was an independent risk factor for overall survival (OS) of GC patients and FTO could promote GC metastasis by upregulating the expression of Integrin β1(ITGB1) via decreasing its m6A level. These results indicated that FTO can be a potent GC biomarker for prognosis prediction as well as a potential target in GC treatment.

Keywords:  FTO; ITGB1; gastric cancer; m6A; metastasis

DOI:  https://doi.org/10.3389/fonc.2021.681280
Oncogene. 2021 Jul 17.

IDH2 contributes to tumorigenesis and poor prognosis by regulating m6A RNA methylation in multiple myeloma.

Sha Song, Gao Fan, Qi Li, Qi Su, Xinyun Zhang, Xiaofeng Xue, Zhiming Wang, Chen'ao Qian, Zhou Jin, Bingzong Li, Wenzhuo Zhuang.

Epigenetic alterations have been previously shown to contribute to multiple myeloma (MM) pathogenesis via DNA methylations and histone modifications. RNA methylation, a novel epigenetic modification, is required for cancer cell survival, and targeting this pathway has been proposed as a new therapeutic strategy. The extent to the N6-methyladenosine (m6A)-regulatory pathway functions in MM remains unknown. Here, we show that an imbalance of RNA methylation may underlies the tumorigenesis of MM. Mechanistically, isocitrate dehydrogenase 2 (IDH2) is highly expressed in CD138+ cells from MM and its levels appear a progressive increase in the progression of plasma cell dyscrasias. Downregulation of IDH2 increases global m6A RNA levels and reduces myeloma cell growth in vitro, decreases the burden of disease and prolongs overall survival in vivo. IDH2 regulates RNA methylation by activating the RNA demethylase FTO, which is an α-KG-dependent dioxygenase. Furthermore, IDH2-mediated FTO activation decreases the m6A level on WNT7B transcripts, then increases WNT7B expression and thus activated Wnt signaling pathway. Moreover, survival analysis indicates that the elevated expression of IDH2 predicts a poor prognosis. Higher expression of FTO is related to higher International Staging System (ISS) stage and higher Revised-ISS (R-ISS) stage of MM. Collectively, our studies reveal that IDH2 regulates global m6A RNA modification in MM via targeting RNA demethylases FTO. The imbalance of m6A methylation activates the Wnt signaling pathway by enhancing the WNT7B expression, and thus promoting tumorigenesis and progression of MM. IDH2 might be used as a therapeutic target and a possible prognostic factor for MM.

DOI: https://doi.org/10.1038/s41388-021-01939-7
Cancer Lett. 2021 Jul 20. pii: S0304-3835(21)00349-9. [Epub ahead of print]

Cigarette smoking induces aberrant N6-methyladenosine of DAPK2 to promote non-small cell lung cancer progression by activating NF-κB pathway.

Meng Jin, Guangcai Li, Wei Liu, Xiaofeng Wu, Jing Zhu, Dong Zhao, Zhaofu Zeng, Mengqing Xiong, Yan Song, Xin He, Qingfeng Zhang, Ke Hu.

  N6-methyladenosine (m6A) has recently emerged as an important regulatory mechanism for gene expression and aberrant m6A modification plays an important role in tumor progression. Emerging evidence has shown that aberrant m6A modification induced by cigarette smoking is involved in carcinogenesis, but whether cigarette smoking affects m6A modification and thus deteriorates to NSCLC is still unclear. Here, we identified a tumor suppressor gene-DAPK2 which was significantly associated with poor prognosis of NSCLC patients, especially in patients with a smoking history. Low levels of DAPK2 were detected in smokers and in NSCLC tissues. Cigarette smoking induced aberrant N6-methyladenosine modification of DAPK2, which resulted in decreased DAPK2 mRNA stability and expression of its mRNA and protein. This modification was mediated by the m6A "writer" METTL3 and the m6A "reader" YTHDF2. Mechanistically, we further demonstrated that DAPK2 functions as a tumor suppressor and downregulation of DAPK2 substantially enhances the proliferation and migration abilities in vitro and in vivo by activating NF-κB signaling pathway. Notably, the BAY 11-7085, a NF-κB signaling selective inhibitor, was shown to efficiently suppressed downregulation of DAPK2-induced oncogenic phenotypes of NSCLC cells. Our study reveals that cigarette smoking induces aberrant N6-methyladenosine of DAPK2 to promote NSCLC progression, which provides new insight into the mechanisms of NSCLC progression and a specific therapeutic target for NSCLC patients with a smoking history.

Keywords:  DAPK2; METTL3; NF-κB; NSCLC; Smoking; YTHDF2; m(6)A

DOI:  https://doi.org/10.1016/j.canlet.2021.07.022
J Transl Med. 2021 Jul 22. 19(1): 316

N6-Methyladenosine RNA modification in cerebrospinal fluid as a novel potential diagnostic biomarker for progressive multiple sclerosis.

Fei Ye, Tianzhu Wang, Xiaoxin Wu, Jie Liang, Jiaoxing Li, Wenli Sheng.

   BACKGROUND: Progressive multiple sclerosis (PMS) is an uncommon and severe subtype of MS that worsens gradually and leads to irreversible disabilities in young adults. Currently, there are no applicable or reliable biomarkers to distinguish PMS from relapsing-remitting multiple sclerosis (RRMS). Previous studies have demonstrated that dysfunction of N6-methyladenosine (m6A) RNA modification is relevant to many neurological disorders. Thus, the aim of this study was to explore the diagnostic biomarkers for PMS based on m6A regulatory genes in the cerebrospinal fluid (CSF).
METHODS: Gene expression matrices were downloaded from the ArrayExpress database. Then, we identified differentially expressed m6A regulatory genes between MS and non-MS patients. MS clusters were identified by consensus clustering analysis. Next, we analyzed the correlation between clusters and clinical characteristics. The random forest (RF) algorithm was applied to select key m6A-related genes. The support vector machine (SVM) was then used to construct a diagnostic gene signature. Receiver operating characteristic (ROC) curves were plotted to evaluate the accuracy of the diagnostic model. In addition, CSF samples from MS and non-MS patients were collected and used for external validation, as evaluated by an m6A RNA Methylation Quantification Kit and by real-time quantitative polymerase chain reaction.
RESULTS: The 13 central m6A RNA methylation regulators were all upregulated in MS patients when compared with non-MS patients. Consensus clustering analysis identified two clusters, both of which were significantly associated with MS subtypes. Next, we divided 61 MS patients into a training set (n = 41) and a test set (n = 20). The RF algorithm identified eight feature genes, and the SVM method was successfully applied to construct a diagnostic model. ROC curves revealed good performance. Finally, the analysis of 11 CSF samples demonstrated that RRMS samples exhibited significantly higher levels of m6A RNA methylation and higher gene expression levels of m6A-related genes than PMS samples.
CONCLUSIONS: The dynamic modification of m6A RNA methylation is involved in the progression of MS and could potentially represent a novel CSF biomarker for diagnosing MS and distinguishing PMS from RRMS in the early stages of the disease.

Keywords:  Cerebrospinal fluid (CSF); Diagnostic biomarker; N6-methyladenosine (m6A); Progressive multiple sclerosis (PMS)

DOI:  https://doi.org/10.1186/s12967-021-02981-5
Front Oncol. 2021 ;11 709579

m6A Regulator-Mediated Methylation Modification Model Predicts Prognosis, Tumor Microenvironment Characterizations and Response to Immunotherapies of Clear Cell Renal Cell Carcinoma.

Wenhao Xu, Xi Tian, Wangrui Liu, Aihetaimujiang Anwaier, Jiaqi Su, Wenkai Zhu, Fangning Wan, Guohai Shi, Gaomeng Wei, Yuanyuan Qu, Hailiang Zhang, Dingwei Ye.

   Background: This study aims to establish an N6-methyladenosine (m6A) RNA methylation regulators-mediated methylation model and explore its role in predicting prognostic accuracy of immune contexture and characterizations of clear cell renal cell carcinoma (ccRCC).
Methods: The m6A modification subclasses (m6AMS) were identified by unsupervised cluster analysis and three clusters were determined by consensus clustering algorithm in a discovering cohort. Testing and real-world validation cohorts were used to identify predictive responses for immune checkpoint therapies (ICTs) of m6AMS.
Results: Prognostic implications landscape of m6A regulators in cancers and its differential expression levels in ccRCC patients were identified. Based on discovering cohort, ccRCC were automatically divided into three m6AMS, and cluster 3 showed significant worse survival than cluster 1/2. Importantly, it was found that the immune checkpoint molecules expression was significantly elevated in cluster 3. Besides, m6A scoreLow group (cluster 1&2) have significantly elevated TIDE score compared with m6A scoreHigh group (cluster 3). There was conspicuous tertiary lymphoid tissue, aggressive phenotype, elevated glycolysis, expression of PD-L1, abundance of CD8+ T cells, CD4+ FOXP3+ Treg cells and TCRn immune cells infiltration in the high m6A score group. Interestingly, there are significantly increased patients with clinical benefit in m6A scoreHigh group in 368 patients receiving ICTs from testing IMvigor210 (n = 292) and validation FUSCC (n = 55) cohorts.
Conclusion: Our discovery highlights the relationship between tumor epigenetic heterogeneity and immune contexture. Immune-rejection cluster 3 has pro-tumorigenic immune infiltration, and shows significant clinical benefits for ccRCC patients receiving ICTs, enabling patient selection for future clinical treatment.

Keywords:  N6-methyladenosine; clear cell renal cell carcinoma; immunotherapies; m6A modification subclasses; tumor microenvironment

DOI:  https://doi.org/10.3389/fonc.2021.709579
Front Cell Dev Biol. 2021 ;9 681745

Comprehensive Analysis of the Immune Infiltrates and PD-L1 of m6A RNA Methylation Regulators in Hepatocellular Carcinoma.

Yangtao Xu, Xiaoqin He, Junjian Deng, Lin Xiong, Yue Li, Xiaoyu Zhang, Wenliang Chen, Xin Liu, Ximing Xu.

  Recently, N 6-methyladenosine (m6A) RNA methylation in eukaryotic mRNA has become increasingly obvious in the pathogenesis and prognosis of cancer. Moreover, tumor microenvironment is involved in the regulation of tumorigenesis. In our research, the clinical data, including 374 tumor and 50 normal patients, were obtained from The Cancer Genome Atlas (TCGA). Then 19 m6A regulators were selected from other studies. Hepatocellular carcinoma (HCC) patients were clustered in cluster1/2, according to the consensus clustering for the m6A RNA regulators. We found that m6A regulators were upregulated in cluster1. The cluster1 was associated with higher programmed death ligand 1 (PD-L1) expression level, higher immunoscore, worse prognosis, and distinct immune cell infiltration compared with cluster2. Five risk signatures were identified, including YTH N6-methyladenosine RNA-binding protein 1, YTHDF2, heterogeneous nuclear ribonucleoprotein C, WT1-associated protein, and methyltransferase-like 3, based on univariate Cox and least absolute shrinkage and selection operator regression analysis. High-risk group and low-risk group HCC patients were selected based on the risk score. Similarly, the high-risk group was extremely associated with higher PD-L1 expression level, higher grade, and worse overall survival (OS). Also, cluster1 was mainly enriched in high-risk group. Receiver operating characteristic (ROC) and a nomogram were used to predict the ability and the probability of 3- and 5-year OS of HCC patients. The time-dependent ROC curve (AUC) reached 0.77, 0.67, and 0.68 at 1, 3, and 5 years in the training dataset. Also, AUC areas of 1, 3, and 5 years were 0.7, 0.63, and 0.55 in the validation dataset. The gene set enrichment analysis showed that MTOR signaling pathway and WNT signaling pathway were correlated with cluster1 and high-risk group. Collectively, the research showed that the m6A regulators were significantly associated with tumor immune microenvironment in HCC. Risk characteristics based on m6A regulators may predict prognosis in patients with HCC and provide a new therapeutic target for improving the efficacy of immunotherapy.

Keywords:  PD-L1; hepatocellular carcinoma; immune infiltrates; m6A RNA methylation; prognosis; tumor immune microenvironment

DOI:  https://doi.org/10.3389/fcell.2021.681745
Front Cell Dev Biol. 2021 ;9 687756

m6A Regulator-Associated Modification Patterns and Immune Infiltration of the Tumor Microenvironment in Hepatocarcinoma.

Jianhao Li, Weiwei Wang, Yubing Zhou, Liwen Liu, Guizhen Zhang, Kelei Guan, Xichun Cui, Xin Liu, Maoxin Huang, Guangying Cui, Ranran Sun.

  Background: Immunotherapy elicits durable responses in many tumors. Nevertheless, the positive response to immunotherapy always depends on the dynamic interactions between the tumor cells and infiltrating lymphocytes in the tumor microenvironment (TME). Currently, the application of immunotherapy in hepatocellular carcinoma (HCC) has achieved limited success. The ectopic modification of N6-methyladenosine (m6A) is a common feature in multiple tumors. However, the relationship between m6A modification with HCC clinical features, prognosis, immune cell infiltration, and immunotherapy efficacy remains unclear. Materials and Methods: Here, we comprehensively evaluated m6A modification clusters based on 22 m6A regulators and systematically explored the relationship between m6A modification with tumor progression, prognosis, and immune cell infiltration characteristics. The m6Ascore was calculated by principal component analysis to quantify the m6A modifications of individual patients. Key regulators involved in immunoregulation in HCC were identified using immunohistochemistry and immunofluorescence. Results: Three distinct m6A modification clusters were identified. The m6A clusters were significantly associated with clinical features, prognosis, and immune cell infiltration. The three clusters were highly consistent with the three tumor immune phenotypes, i.e., immune-excluded, immune-inflamed, and immune-desert. Comprehensive bioinformatics analysis revealed that high m6Ascore was closely associated with tumor progression, poor prognosis, and immunotherapy non-response. m6A regulators were dysregulated in HCC tissues. Hence, they play a role as predictors of poor prognosis. Tissue microarray demonstrated that overexpressed YTHDF1 was associated with low CD3+ and CD8+ T cell infiltration in HCC. Conclusion: Our findings demonstrate that m6A modification patterns play a crucial role in the tumor immune microenvironment and the prognosis of HCC. High YTHDF1 expression is closely associated with low CD3+ and CD8+ T cell infiltration in HCC.

Keywords:  N6-methyladenosine; hepatocellular carcinoma; immune infiltration; prognosis; tumor microenvironment

DOI:  https://doi.org/10.3389/fcell.2021.687756
Pathog Dis. 2021 Jul 19. pii: ftab036. [Epub ahead of print]

Knockdown of METTL3 inhibits enterovirus 71 induced apoptosis of mouse Schwann cell through regulation of autophagy.

Yulian Xiao, Yanping Yang, Dandan Hu.

  Enterovirus A71 (EV-A71 or EV-71) is an RNA virus which cause hand, foot and mouse disease (HFMD) in children. The N6-methyladenosine (m6A) of RNA is a common RNA modification involved in various biological events. METTL3 is an m6A methyltransferase which regulates EV-71 replication. EV-71 infection induces autophagy, which promotes EV-71 replication too. In this study, we explored the role of METTL3 in EV-71 infection-induced autophagy. We constructed lentivirus expressing METTL3 specific shRNA and knocked down the endogenous METTL3 in mouse Schwann cells. We infected normal Schwann cells and METTL3 knock down Schwann cells and compared the viral titer, expression of autophagy-related proteins and apoptosis-related protein. Transduction of lentivirus expressing METTL3 shRNA significantly decreased the endogenous METTL3. Knocking down METTL3 decreased the viral titer of EV-71 after infection. Knocking down METTL3 prevented EV-71 induced cell death and suppressed EV-71-induced expression of Bax while rescued Bcl-2 expression after EV-71 infection. Knocking down METTL3 inhibited EV-71-induced expression of Atg5, Atg7 and LC3 II. Knocking down METTL3 inhibited EV-71 induced apoptosis and autophagy. In summary, our study described the relation of METTL3 and autophagy during EV-71 infection.

Keywords:  METTL3; apoptosis; autophagy; enterovirus 71

DOI:  https://doi.org/10.1093/femspd/ftab036
Trends Genet. 2021 Jul 19. pii: S0168-9525(21)00170-0. [Epub ahead of print]

Crosstalk between epitranscriptomic and epigenetic mechanisms in gene regulation.

Ryan L Kan, Jianjun Chen, Tamer Sallam.

  Epigenetic modifications occur on genomic DNA and histones to influence gene expression. More recently, the discovery that mRNA undergoes similar chemical modifications that powerfully impact transcript turnover and translation adds another layer of dynamic gene regulation. Central to precise and synchronized regulation of gene expression is intricate crosstalk between multiple checkpoints involved in transcript biosynthesis and processing. There are more than 100 internal modifications of RNA in mammalian cells. The most common is N6-methyladenosine (m6A) methylation. Although m6A is established to influence RNA stability dynamics and translation efficiency, rapidly accumulating evidence shows significant crosstalk between RNA methylation and histone/DNA epigenetic mechanisms. These interactions specify transcriptional outputs, translation, recruitment of chromatin modifiers, as well as the deployment of the m6A methyltransferase complex (MTC) at target sites. In this review, we dissect m6A-orchestrated feedback circuits that regulate histone modifications and the activity of regulatory RNAs, such as long noncoding (lnc)RNA and chromosome-associated regulatory RNA. Collectively, this body of evidence suggests that m6A acts as a versatile checkpoint that can couple different layers of gene regulation with one another.

Keywords:  RNA methylation; RNA modification; epigenetics; gene regulation

DOI:  https://doi.org/10.1016/j.tig.2021.06.014
Mol Cell. 2021 Jul 20. pii: S1097-2765(21)00503-7. [Epub ahead of print]

m6A RNA methylation regulates promoter- proximal pausing of RNA polymerase II.

Junaid Akhtar, Yoan Renaud, Steffen Albrecht, Yad Ghavi-Helm, Jean-Yves Roignant, Marion Silies, Guillaume Junion.

  RNA polymerase II (RNAP II) pausing is essential to precisely control gene expression and is critical for development of metazoans. Here, we show that the m6A RNA modification regulates promoter-proximal RNAP II pausing in Drosophila cells. The m6A methyltransferase complex (MTC) and the nuclear reader Ythdc1 are recruited to gene promoters. Depleting the m6A MTC leads to a decrease in RNAP II pause release and in Ser2P occupancy on the gene body and affects nascent RNA transcription. Tethering Mettl3 to a heterologous gene promoter is sufficient to increase RNAP II pause release, an effect that relies on its m6A catalytic domain. Collectively, our data reveal an important link between RNAP II pausing and the m6A RNA modification, thus adding another layer to m6A-mediated gene regulation.

Keywords:  RNA modification; RNA polymerase II pausing; m(6)A; transcription elongation; transcriptional checkpoint

DOI:  https://doi.org/10.1016/j.molcel.2021.06.023
J Cell Mol Med. 2021 Jul 21.

Conditional deficiency of m6A methyltransferase Mettl14 in substantia nigra alters dopaminergic neuron function.

Yan Teng, Zhihao Liu, Xingmin Chen, Yanzhuo Liu, Fan Geng, Weidong Le, Haisong Jiang, Lu Yang.

  N6-Methyladenosine (m6A) is the most prevalent internal modification in messenger RNAs (mRNAs) of eukaryotes and plays a vital role in post-transcriptional regulation. Recent studies demonstrated that m6A is essential for the normal function of the central nervous system (CNS), and the deregulation of m6A leads to a series of CNS diseases. However, the functional consequences of m6A deficiency within the dopaminergic neurons of adult brain are elusive. To evaluate the necessity of m6A in dopaminergic neuron functions, we conditionally deleted Mettl14, one of the most important part of m6A methyltransferase complexes, in the substantia nigra (SN) region enriched with dopaminergic neurons. By using rotarod test, pole test, open-field test and elevated plus maze, we found that the deletion of Mettl14 in the SN region induces impaired motor function and locomotor activity. Further molecular analysis revealed that Mettl14 deletion significantly reduced the total level of m6A in the mRNA isolated from SN region. Tyrosine hydroxylase (TH), an essential enzyme for dopamine synthesis, was also down-regulated upon Mettl14 deletion, while the activation of microglia and astrocyte was enhanced. Moreover, the expression of three essential transcription factors in the regulation of TH including Nurr1, Pitx3 and En1, with abundant m6A-binding sites on their RNA 3'-untranslated regions (UTR), was significantly decreased upon Mettl14 deletion in SN. Our finding first confirmed the significance of m6A in maintaining normal dopaminergic function in the SN of adult mouse.

Keywords:  Mettl14; dopaminergic neuron; m6A; tyrosine hydroxylase

DOI:  https://doi.org/10.1111/jcmm.16740
Bioengineered. 2021 Dec;12(1): 4081-4091

N6-methyladenosine (m6A)-mediated up-regulation of long noncoding RNA LINC01320 promotes the proliferation, migration, and invasion of gastric cancer via miR495-5p/RAB19 axis.

Naijun Hu, Hong Ji.

  Gastric cancer is one of the most common malignant tumors. Long non-coding RNAs play crucial roles in gastric cancer progression. This study investigated the effect of LINC01320 on malignant behaviors of gastric cancer cells and explored its possible molecular mechanism. LINC01320 expression in gastric cancer tissues and cell lines was measured by qRT-PCR. Cell proliferation, transwell, and cell cloning assays were used to detect the effect of LINC01320 on the proliferation, migration, and invasion abilities, respectively, of gastric cancer cells. Bioinformatics analysis was used to predict the binding of miR-495-5p with LINC01320 and RAB19. A luciferase reporter assay was performed to verify their interactions. Finally, the N6-methyladenosine (m6A) modification of LINC01320 by METTL14 was identified through RIP experiments. LINC01320 was highly expressed in gastric cancer tissues and cells. LINC01320 overexpression promoted the proliferation, migration, and invasion of gastric cancer cells, while LINC01320 knockdown exerted the opposite effects. Moreover, miR-495-5p was predicted and demonstrated to target LINC01320 and RAB19. LINC01320 sponged miR-495-5p to regulate the expression of RAB19. Additionally, LINC01320-induced increases in cell viability, migration, and invasion of gastric cancer were alleviated by miR-495-5p and silenced RAB19. Furthermore, epigenetic studies showed that METTL14-mediated m6A modification led to LINC01320 up-regulation. METTL14 regulated the m6A modification of LINC01320. Overexpressed LINC01320 contributed to the aggressive phenotype of gastric cancer cells via regulating the miR-495-5p/RAB19 axis. This finding may provide new potential targets for treating gastric cancer.

Keywords:  LncRNA linc01320; gastric cancer; miR-495-5p; n6-methyladenosine; rab19

DOI:  https://doi.org/10.1080/21655979.2021.1953210
Front Cell Dev Biol. 2021 ;9 658642

HNRNPA2B1 Affects the Prognosis of Esophageal Cancer by Regulating the miR-17-92 Cluster.

Kexin Li, Jiongyu Chen, Xiaoying Lou, Yiling Li, Benheng Qian, Danfei Xu, Yue Wu, Shaohui Ma, Donghong Zhang, Wei Cui.

  N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotes. Accumulating evidence suggests that dysregulation of m6A modification significantly correlates with tumorigenesis and progression. In this study, we observed an increased expression and positive correlations of all 25 m6A regulators in esophageal cancer (ESCA) data obtained from the TCGA database. Through expression profiling of these regulators, a prognostic score model containing HNRNPA2B1, ALKBH5, and HNRNPG was established, and the high-risk subgroup exhibited strong positive correlations with ESCA progression and outcome. The risk score obtained from this model may represent an independent predictor of ESCA prognosis. Notably, the gene most frequently associated with increased risk was HNRNPA2B1; in ESCA, the increased expression of this gene alone predicted poor prognosis by affecting tumor-promoting signaling pathways through miR-17-92 cluster. An experimental study demonstrated that elevated HNRNPA2B1 expression was positively associated with distant metastasis and lymph node stage, and predicted the poor outcomes of ESCA patients. Knockdown of HNRNPA2B1 significantly decreased the expression of miR-17, miR-18a, miR-20a, miR-93, and miR-106b and inhibited the proliferation of ESCA cells. Therefore, our study indicated that the dynamic changes in 25 m6A regulators were associated with the clinical features and prognosis of patients with ESCA. Importantly, HNRNPA2B1 alone may affect the prognosis of patients with ESCA by regulating the miR-17-92 cluster.

Keywords:  HNRNPA2B1; esophageal cancer; m6A; miR-17-92 cluster; prognosis

DOI:  https://doi.org/10.3389/fcell.2021.658642
Cancer Res. 2021 Jul 23. pii: canres.0468.2021. [Epub ahead of print]

M6A Demethylase ALKBH5 Regulates PD-L1 Expression and Tumor Immunoenvironment in Intrahepatic Cholangiocarcinoma.

Xinyao Qiu, Shuai Yang, Shan Wang, Jianmin Wu, Bo Zheng, Kaiting Wang, Siyun Shen, Seogsong Jeong, Zhixuan Li, Yanjing Zhu, Tong Wu, Xuan Wu, Rui Wu, Weiwei Liu, Hong-Yang Wang, Lei Chen.

N6-methyladenosine (m6A) has been reported as an important mechanism of post-transcriptional regulation. Programmed death-ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. Here we report ALKBH5 as an important m6A demethylase that orchestrates PD-L1 expression in intrahepatic cholangiocarcinoma (ICC). Regulation of PD-L1 expression by ALKBH5 was confirmed in human ICC cell lines. Sequencing of the m6A methylome identified PD-L1 mRNA as a direct target of m6A modification whose levels were regulated by ALKBH5. Furthermore, ALKBH5 and PD-L1 mRNA were shown to interact. ALKBH5 deficiency enriched m6A modification in the 3'UTR region of PD-L1 mRNA, thereby promoting its degradation in a YTHDF2-dependent manner. In vitro and in vivo, tumor-intrinsic ALKBH5 inhibited the expansion and cytotoxicity of T cells by sustaining tumor cell PD-L1 expression. The ALKBH5-PD-L1-regulating axis was further confirmed in human ICC specimens. Single-cell mass cytometry analysis unveiled a complex role of ALKBH5 in the tumor immune microenvironment by promoting the expression of PD-L1 on monocytes/macrophages and decreasing the infiltration of myeloid-derived suppressor-like cells. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with strong nuclear expression patterns of ALKBH5 are more sensitive to anti-PD1 immunotherapy. Collectively, these results describe a new regulatory mechanism of PD-L1 by mRNA epigenetic modification by ALKBH5 and the potential role of ALKBH5 in immunotherapy response, which might provide insights for cancer immunotherapies.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0468
Front Mol Biosci. 2021 ;8 665222

Identification of the m6A RNA Methylation Regulators WTAP as a Novel Prognostic Biomarker and Genomic Alterations in Cutaneous Melanoma.

Zi-Yi Feng, Ting Wang, Xin Su, Shu Guo.

  Background: The purpose of our research was to establish a gene signature and determine the prognostic value of m6A methylation regulators in cutaneous melanoma and WTAP as a protective gene in cutaneous melanoma prognosis, we also evaluated gene mutations in cutaneous melanoma. Methods: We downloaded the RNA-seq transcriptome data and the clinical information for cutaneous melanoma patients from the GTEx and TCGA databases. Consensus clustering analysis was applied to divide the samples into two groups. Then the least absolute shrinkage and selection operator (LASSO) analyses were conducted to construct a risk signature, and we use external and internal datasets to verify its predictive value. We further searched the cBioPortal tools to detect genomic alterations and WTAP mutations. Finally, WTAP was further identified as a prognostic factor, and the related mechanisms mediated by WTAP were predicted by gene set enrichment analysis (GSEA). Experimental validations and have been further carried out. Results: Notably, m6A RNA methylation regulators play significant roles in tumorigenesis and development. In total, we selected three subtypes of cutaneous melanoma according to consensus clustering of the m6A RNA methylation regulators, and the stage of cutaneous melanoma was proven to be related to the subtypes. The Cox regression and LASSO analyses built a risk signature including ELF3, ZC3H13 and WTAP. The prognostic value of the risk signature in internal and external datasets have been proven then. The whole-genome and selected gene WTAP mutations were further explored. WTAP as a single prognostic factor was also explored and found to serve as an independent protective prognostic factor. Conclusions: Our study constructed a stable risk signature composed of m6A RNA methylation regulators in cutaneous melanoma. Moreover, WTAP was identified as a valuable prognostic factor and potential molecular target for cutaneous melanoma treatment.

Keywords:  WTAP; biomarker; cutaneous melanoma; genomic alterations; m6A regulators; survival analysis

DOI:  https://doi.org/10.3389/fmolb.2021.665222
J Inflamm Res. 2021 ;14 3289-3306

The Emerging Clinical Application of m6A RNA Modification in Inflammatory Bowel Disease and Its Associated Colorectal Cancer.

Xinwei Xu, Jintu Huang, Dickson Kofi Wiredu Ocansey, Yuxuan Xia, Zihan Zhao, Zhiwei Xu, Yongmin Yan, Xu Zhang, Fei Mao.

  Methylation, first proposed in DNAs, but later found in RNAs, serves as one of the most widespread epigenetic modifications in eukaryotes, where N6-methyladenosine (m6A) modification has been found to play an important role in a variety of cancers including colorectal cancer (CRC). Under the action of various enzymes and proteins, the regulatory role of m6A in RNAs and immune cells has also been gradually realized. This paper reviews the general biogenesis and effects of m6A, and its emerging crucial role in intestinal mucosal immunity via the regulation of RNAs and immune cells, and thus closely related to the occurrence and development of inflammatory bowel disease (IBD) and CRC. m6A-related genes and regulatory factors are expected to be potential predictive markers and therapeutic targets.

Keywords:  N6-methyladenosine; colorectal cancer; inflammatory bowel disease; intestinal mucosal immunity; non-coding RNA

DOI:  https://doi.org/10.2147/JIR.S320449
Proc Natl Acad Sci U S A. 2021 Jul 27. pii: e2104805118. [Epub ahead of print]118(30):

Widespread remodeling of the m6A RNA-modification landscape by a viral regulator of RNA processing and export.

Kalanghad Puthankalam Srinivas, Daniel P Depledge, Jonathan S Abebe, Stephen A Rice, Ian Mohr, Angus C Wilson.

  N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.

Keywords:  N6-methyladenosine; RNA modification; direct RNA sequencing; herpes simplex virus; nanopore

DOI:  https://doi.org/10.1073/pnas.2104805118
Nat Metab. 2021 Jul;3(7): 940-953

Transcriptional regulation of N6-methyladenosine orchestrates sex-dimorphic metabolic traits.

David A Salisbury, David Casero, Zhengyi Zhang, Dan Wang, Jason Kim, Xiaohui Wu, Laurent Vergnes, Aashiq H Mirza, Paola Leon-Mimila, Kevin J Williams, Adriana Huertas-Vazquez, Samie R Jaffrey, Karen Reue, Jianjun Chen, Tamer Sallam.

Males and females exhibit striking differences in the prevalence of metabolic traits including hepatic steatosis, a key driver of cardiometabolic morbidity and mortality. RNA methylation is a widespread regulatory mechanism of transcript turnover. Here, we show that presence of the RNA modification N6-methyladenosine (m6A) triages lipogenic transcripts for degradation and guards against hepatic triglyceride accumulation. In male but not female mice, this protective checkpoint stalls under lipid-rich conditions. Loss of m6A control in male livers increases hepatic triglyceride stores, leading to a more 'feminized' hepatic lipid composition. Crucially, liver-specific deletion of the m6A complex protein Mettl14 from male and female mice significantly diminishes sex-specific differences in steatosis. We further surmise that the m6A installing machinery is subject to transcriptional control by the sex-responsive BCL6-STAT5 axis in response to dietary conditions. These data show that m6A is essential for precise and synchronized control of lipogenic enzyme activity and provide insights into the molecular basis for the existence of sex-specific differences in hepatic lipid traits.

DOI: https://doi.org/10.1038/s42255-021-00427-2
J Hematol Oncol. 2021 Jul 17. 14(1): 112

LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC.

Xinyi Qian, Juze Yang, Qiongzi Qiu, Xufan Li, Chengxi Jiang, Jia Li, Liangliang Dong, Kejing Ying, Bingjian Lu, Enguo Chen, Pengyuan Liu, Yan Lu.

   BACKGROUND: Long non-coding RNAs (lncRNAs) are important epigenetic regulators, which play critical roles in diverse physiological and pathological processes. However, the regulatory mechanism of lncRNAs in lung carcinogenesis remains elusive. Here, we characterized a novel oncogenic lncRNA, designated as Lung Cancer Associated Transcript 3 (LCAT3).
METHODS: We predicted and validated LCAT3 by analyzing RNA-sequencing (RNA-seq) data of lung cancer tissues from TCGA. Methylated RNA immunoprecipitation was performed to assess m6A modification on LCAT3. The LCAT3-FUBP1-MYC axis was assessed by dual-luciferase reporter, RNA immunoprecipitation and Chromatin immunoprecipitation assays. Signaling pathways altered by LCAT3 knockdown were identified using RNA-seq. Furthermore, the mechanism of LCAT3 was investigated using loss-of-function and gain-of-function assays in vivo and in vitro.
RESULTS: LCAT3 was found to be up-regulated in lung adenocarcinomas (LUAD), and its over-expression was associated with the poor prognosis of LUAD patients. LCAT3 upregulation is attributable to N6-methyladenosine (m6A) modification mediated by methyltransferase like 3 (METTL3), leading to LCAT3 stabilization. Biologically, loss-of-function assays revealed that LCAT3 knockdown significantly suppressed lung cancer cell proliferation, migration and invasion in vitro, and inhibited tumor growth and metastasis in vivo. LCAT3 knockdown induced cell cycle arrest at the G1 phase. Mechanistically, LCAT3 recruited Far Upstream Element Binding Protein 1 (FUBP1) to the MYC far-upstream element (FUSE) sequence, thereby activating MYC transcription to promote proliferation, survival, invasion and metastasis of lung cancer cells.
CONCLUSIONS: Taken together, we identified and characterized LCAT3 as a novel oncogenic lncRNA in the lung, and validated the LCAT3-FUBP1-MYC axis as a potential therapeutic target for LUAD.

Keywords:  FUBP1; LCAT3; Long noncoding RNAs; Lung cancer; MYC; N6-methyladenosine

DOI:  https://doi.org/10.1186/s13045-021-01123-0
Front Immunol. 2021 ;12 705232

m6A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection.

Zijie Xia, Jihao Xu, Eugene Lu, Wei He, Silu Deng, Ai-Yu Gong, Juliane Strass-Soukup, Gislaine A Martins, Guoqing Lu, Xian-Ming Chen.

  Increasing evidence supports that N6-methyladenosine (m6A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m6A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m6A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m6A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m6A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m6A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.

Keywords:  ALKBH5; Cryptosporidium; Igtp; Irgm2; RNA stability; defense; intestinal epithelium; m6A

DOI:  https://doi.org/10.3389/fimmu.2021.705232
Front Mol Biosci. 2021 ;8 682471

Hub Long Noncoding RNAs with m6A Modification for Signatures and Prognostic Values in Kidney Renal Clear Cell Carcinoma.

Gaoteng Lin, Huadong Wang, Yuqi Wu, Keruo Wang, Gang Li.

  Background: N6-methyladenosine (m6A)-modified long noncoding RNAs (m6A-lncRNAs) have been proven to be involving in regulating tumorigenesis, invasion, and metastasis for a variety of tumors. The present study aimed to screen lncRNAs with m6A modification and investigate their biological signatures and prognostic values in kidney renal clear cell carcinoma (KIRC). Materials and Methods: lncRNA-seq, miRNA-seq, and mRNA-seq profiles of KIRC samples and the clinical characteristics of corresponding patients were downloaded from The Cancer Genome Atlas (TCGA). The R package "edgeR" was utilized to perform differentially expressed analysis on these profiles to gain DElncRNAs, DEmiRNAs, and DEmRNAs, respectively. The results of intersection of DElncRNAs and m6A-modified genes were analyzed by the weighted gene co-expression network analysis (WGCNA) to screen hub m6A-lncRNAs. Then, WGCNA was also used to construct an lncRNA-miRNA-mRNA (ceRNA) network. The Cox regression analysis was conducted on hub m6A-lncRNAs to construct the m6A-lncRNAs prognostic index (m6AlRsPI). Receiver operating characteristic (ROC) curve was used to assess the predictive ability of m6AlRsPI. The m6AlRsPI model was tested by internal and external cohorts. The molecular signatures and prognosis for hub m6A-lncRNAs and m6AlRsPI were analyzed. The expression level of hub m6A-lncRNAs in KIRC cell lines were quantified by qRT-PCR. Results: A total of 21 hub m6A-lncRNAs associated with tumor metastasis were identified in the light of WGCNA. The ceRNA network for 21 hub m6A-lncRNAs was developed. The Cox regression analysis was performed on the 21 hub m6A-lncRNAs, screening two m6A-lncRNAs regarded as independent prognostic risk factors. The m6AlRsPI was established based on the two m6A-lncRNAs as follows: (0.0006066 × expression level of LINC01820) + (0.0020769 × expression level of LINC02257). The cutoff of m6AlRsPI was 0.96. KM survival analysis for m6AlRsPI showed that the high m6AlRsPI group could contribute to higher mortality. The area under ROC curve for m6AlRsPI for predicting 3- and 5-year survival was 0.760 and 0.677, respectively, and the m6AlRsPI was also tested. The mutation and epithelial-mesenchymal transition (EMT) analysis for m6AlRsPI showed that the high m6AIRsPI group had more samples with gene mutation and had more likely caused EMT. Finally, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for mRNAs interacted with the two m6A-lncRNAs, showing they were involved in the process of RNA splicing and regulation of the mRNA surveillance pathway. qRT-PCR analysis showed that the two m6A-lncRNAs were upregulated in KIRC. Conclusion: In the present study, hub m6A-lncRNAs were determined associated with metastasis in KIRC, and the ceRNA network demonstrated the potential carcinogenic regulatory pathway. Two m6A-lncRNAs associated with the overall survival were screened and m6AlRsPI was constructed and validated. Finally, the molecular signatures for m6AlRsPI and the two m6A-lncRNAs were analyzed to investigate the potential modulated processes in KIRC.

Keywords:  N6-methyladenosine; WGCNA; kidney renal clear cell carcinoma; long noncoding RNA; prognostic index

DOI:  https://doi.org/10.3389/fmolb.2021.682471
Nat Biotechnol. 2021 Jul 19.

Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore.

Ploy N Pratanwanich, Fei Yao, Ying Chen, Casslynn W Q Koh, Yuk Kei Wan, Christopher Hendra, Polly Poon, Yeek Teck Goh, Phoebe M L Yap, Jing Yuan Chooi, Wee Joo Chng, Sarah B Ng, Alexandre Thiery, W S Sho Goh, Jonathan Göke.

RNA modifications, such as N6-methyladenosine (m6A), modulate functions of cellular RNA species. However, quantifying differences in RNA modifications has been challenging. Here we develop a computational method, xPore, to identify differential RNA modifications from nanopore direct RNA sequencing (RNA-seq) data. We evaluate our method on transcriptome-wide m6A profiling data, demonstrating that xPore identifies positions of m6A sites at single-base resolution, estimates the fraction of modified RNA species in the cell and quantifies the differential modification rate across conditions. We apply xPore to direct RNA-seq data from six cell lines and multiple myeloma patient samples without a matched control sample and find that many m6A sites are preserved across cell types, whereas a subset exhibit significant differences in their modification rates. Our results show that RNA modifications can be identified from direct RNA-seq data with high accuracy, enabling analysis of differential modifications and expression from a single high-throughput experiment.

DOI: https://doi.org/10.1038/s41587-021-00949-w
Mol Brain. 2021 Jul 19. 14(1): 119

The role m6A RNA methylation is CNS development and glioma pathogenesis.

Ting Pan, Fan Wu, Liwen Li, Shiyan Wu, Fang Zhou, Ping Zhang, Caixing Sun, Liang Xia.

  Epigenetic abnormalities play a crucial role in many tumors, including glioma. RNA methylation occurs as an epigenetic modification similar to DNA methylation and histone modification. m6A methylation is the most common and most intensively studied RNA methylation, which can be found throughout the RNA life cycle and exert biological functions by affecting RNA metabolism. The m6A modification is primarily associated with three types of protease, which are encoded by the writer, eraser and reader genes, respectively. It has been shown that the m6A methylation has close connections with the occurrence and development of many tumors, including glioma. In this study, the concept and the research progress of m6A methylation are reviewed, especially the role of m6A methylation in glioma. Moreover, we will discuss how glioma is paving the way to the development of new therapeutic options based on the inhibition of m6A deposition.

Keywords:  ALKBH5; FTO; GBM; Glioma; METTL3; WTAP; m6A methylation

DOI:  https://doi.org/10.1186/s13041-021-00831-5
J Cell Mol Med. 2021 Jul 21.

Prognostic signature and immune efficacy of m1 A-, m5 C- and m6 A-related regulators in cutaneous melanoma.

Xian Rui Wu, Zheng Chen, Yang Liu, Zi Zi Chen, Fengjie Tang, Zhi Zhao Chen, Jing Jing Li, Jun Lin Liao, Ke Cao, Xiang Chen, Jianda Zhou.

  Cutaneous melanoma (CM) is an aggressive cancer; given that initial and specific signs are lacking, diagnosis is often late and the prognosis is poor. RNA modification has been widely studied in tumour progression. Nevertheless, little progress has been made in the signature of N1 -methyladenosine (m1 A), 5-methylcytosine (m5 C), N6 -methyladenosine (m6 A)-related regulators and the tumour microenvironment (TME) cell infiltration in CM. Our study identified the characteristics of m1 A-, m5 C- and m6 A-related regulators based on 468 CM samples from the public database. Using univariate, multivariate and LASSO Cox regression analysis, a risk model of regulators was established and validated by a nomogram on independent prognostic factors. The gene set variation analysis (GSVA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) clarified the involved functional pathways. A combined single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT approach revealed TME of regulator-related prognostic signature. The nine-gene signature stratified the patients into distinct risk subgroups for personalized prognostic assessment. Additionally, functional enrichment, immune infiltration and immunotherapy response analysis indicated that the high-risk group was correlated with T-cell suppression, while the low-risk group was more sensitive to immunotherapy. The findings presented here contribute to our understanding of the TME molecular heterogeneity in CM. Nine m1 A-, m5 C- and m6 A-related regulators may also be promising biomarkers for future research.

Keywords:  cutaneous melanoma; immunotherapy; m1A; m5C; m6A; prognosis; tumour microenvironment

DOI:  https://doi.org/10.1111/jcmm.16800
Leuk Res. 2021 Jun 29. pii: S0145-2126(21)00154-5. [Epub ahead of print]110 106653

MicroRNA-211 attenuates cell proliferation in T-cell lymphoblastic lymphoma through targeting TCF12.

Licai An, Xijing Li, Jing Yang.

  T-cell lymphoblastic lymphoma (T-LBL) is a class of highly aggressive hematologic neoplasms originating from progenitor T-lymphocytes. MicroRNA (miRNA) is an endogenous RNA molecule with 22 nucleotides in length. Accumulated evidence suggests that miRNA functions as a key regulator in human cancer. Herein, by in silico analysis, we found that miR-211 was a decreased miRNA in T-LBL in high-throughput sequencing data, which was subsequently verified in our cohort. Low miR-211 was closely correlated with bulky disease, high ann arbor stage, relapse and poor prognosis. miR-211 was regulated by N6-methyladenosine (m6A) modification, specifically, m6A methyltransferase METTL14 methylated primary miR-211 (pri-miR-211), expediting pri-miR-211 processing via recruiting DGCR8. Functionally, miR-211 overexpression significantly reduced T-LBL cell viability, DNA synthesis rate and spheroid formation ability, whereas silencing of miR-211 had the opposite effects. In addition, we established the xenograft tumor model and found that miR-211 remarkably inhibited tumor growth in vivo. Further, TCF12 was the direct target of miR-211, miR-211 bound to TCF12 mRNA 3`-untranslated region (UTR) and increased its decay, overexpression of TCF12 could effectively rescue the weakened malignant behavior of T-LBL cells caused by miR-211 overexpression. Collectively, our data clearly demonstrate that miR-211 is a novel tumor suppressor in T-LBL, targeting of miR-211/TCF12 axis may be a potential treatment for T-LBL patients.

Keywords:  N6-methyladenosine; Prognosis; T-cell lymphoblastic lymphoma; miR-211; miRNA processing

DOI:  https://doi.org/10.1016/j.leukres.2021.106653
RNA Biol. 2021 Jul 21. 1-11

RNA 5-methylcytosine modification and its emerging role as an epitranscriptomic mark.

Yaqi Gao, Jingyuan Fang.

  5-methylcytosine (m5C) is identified as an abundant and conserved modification in various RNAs, including tRNAs, mRNAs, rRNAs, and other non-coding RNAs. The application of high-throughput sequencing and mass spectrometry allowed for the detection of m5C at a single-nucleotide resolution and at a global abundance separately; this contributes to a better understanding of m5C modification and its biological functions. m5C modification plays critical roles in diverse aspects of RNA processing, including tRNA stability, rRNA assembly, and mRNA translation. Notably, altered m5C modifications and mutated RNA m5C methyltransferases are associated with diverse pathological processes, such as nervous system disorders and cancers. This review may provide new sights of molecular mechanism and functional importance of m5C modification.

Keywords:  5-methylcytosine (m5C); RNA m5C methyltransferase; RNA methylation; epitranscriptome

DOI:  https://doi.org/10.1080/15476286.2021.1950993
J Chemother. 2021 Jul 21. 1-12

miR-302a-3p suppresses melanoma cell progression via targeting METTL3.

Xiao Chang, Yu-Ying Lin, Li-Na Bai, Wei Zhu.

  The miRNA-302 family plays a critical role in carcinogenesis. As an enzyme that regulates the N6-methyladenosine modification, methyltransferase-like 3 (METTL3) plays important roles in the development and progression of various tumours. However, the upstream regulatory mechanisms of METTL3 in melanoma have not yet been fully investigated. Herein, we investigated the functions of miR-302a-3p and its target RNA METTL3 on proliferation, apoptosis, and invasion of melanoma. Quantitative real-time PCR and immunofluorescence staining were used to measure the expression of METTL3 mRNA and protein level after transfection. miR-302a-3p expression was determined by quantitative reverse transcription-PCR. The cell proliferation, cell cycle progression, apoptosis, colony formation, migration, and cell invasion ability were determined using MTT assay, propidium iodide (PI) staining, Annexin V/PI flow cytometry, plate clone assay, and Transwell migration and invasion assays, respectively. Melanoma cell metastasis was also evaluated using an in vivo model. The effect of METTL3 on the phosphorylation of PI3K and AKT was measured with western blot analysis. Our results showed that miR-302a-3p was significantly downregulated in melanoma and exerted a tumour suppressive role against melanoma progression. We identified METTL3 as a direct target of miR-302a-3p in melanoma cells using bioinformatics analysis and luciferase assay. Furthermore, the enforced overexpression of METTL3 promoted the proliferation, cell cycle progression, cell invasion, migration, expression of epithelial-to-mesenchymal transition markers, and the PI3K-AKT signalling pathway as well as suppressed the apoptosis of melanoma cells. Meanwhile, silencing the expression of METTL3 with specific shRNA demonstrated reverse outcomes of the above phenotypes in melanoma cells. By rescue experiments, we found that the restoration of METTL3 expression in miR-302a-3p-overexpressing melanoma cells successfully recovered the miR-302a-3p-mediated melanoma suppression. The in vivo results also showed that miR-302a-3p substantially inhibited melanoma cell growth and metastasis. In summary, this study demonstrated that miR-302a-3p targets METTL3 and plays tumour suppressive roles in the proliferation, apoptosis, invasion, and migration of melanoma cells.

Keywords:  METTL3; carcinogenesis; m6A; melanoma; miR-302a-3p

DOI:  https://doi.org/10.1080/1120009X.2021.1953886
Insect Mol Biol. 2021 Jul 22.

Active genic machinery for epigenetic RNA modifications in bees.

L Bataglia, Z L P Simões, F M F Nunes.

  Epitranscriptomics is an emerging field of investigation dedicated to the study of post-transcriptional RNA modifications. RNA methylations regulate RNA metabolism and processing, including changes in response to environmental cues. Although RNA modifications are conserved from bacteria to eukaryotes, there is little evidence of a epitranscriptomic pathway in insects. Here we identified genes related to RNA m6 A (N6-methyladenine) and m5 C (5-methylcytosine) methylation machinery in seven bee genomes (Apis mellifera, Melipona quadrifasciata, Frieseomelitta varia, Eufriesea mexicana, Bombus terrestris, Megachile rotundata and Dufourea novaeangliae). In A. mellifera, we validated the expression of methyltransferase genes and found that the global levels of m6 A and m5 C measured in the fat body and brain of adult workers differ significantly. Also, m6 A levels were differed significantly mainly between the fourth larval instar of queens and workers. Moreover, we found a conserved m5 C site in the honeybee 28S rRNA. Taken together, we confirm the existence of epitranscriptomic machinery acting in bees and open avenues for future investigations on RNA epigenetics in a wide spectrum of hymenopteran species. This article is protected by copyright. All rights reserved.

Keywords:  Apis mellifera; RNA methylation; bee; epitranscripitomics; m5C; m6A

DOI:  https://doi.org/10.1111/imb.12726
Cell Death Dis. 2021 Jul 23. 12(8): 732

RBM15-mediated N6-methyladenosine modification affects COVID-19 severity by regulating the expression of multitarget genes.

Yuting Meng, Qiong Zhang, Kaihang Wang, Xujun Zhang, Rongwei Yang, Kefan Bi, Wenbiao Chen, Hongyan Diao.

Severe coronavirus disease 2019 (COVID-19) is characterized by symptoms of lymphopenia and multiorgan damage, but the underlying mechanisms remain unclear. To explore the function of N6-methyladenosine (m6A) modifications in COVID-19, we performed microarray analyses to comprehensively characterize the m6A epitranscriptome. The results revealed distinct global m6A profiles in severe and mild COVID-19 patients. Programmed cell death and inflammatory response were the major biological processes modulated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Further, RBM15, a major m6A methyltransferase, was significantly elevated and positively correlated with disease severity. Silencing RBM15 drastically reduced lymphocyte death in vitro. Knockdown of RBM15 remarkably suppressed the expression levels of multitarget genes related to programmed cell death and inflammatory response. This study shows that SARS-CoV-2 infection alters the m6A epitranscriptome of lymphocytes, particularly in the case of severe patients. RBM15 regulated host immune response to SARS-CoV-2 by elevating m6A modifications of multitarget genes. These findings indicate that RBM15 can serve as a target for the treatment of COVID-19.

DOI: https://doi.org/10.1038/s41419-021-04012-z
Cancers (Basel). 2021 Jul 05. pii: 3372. [Epub ahead of print]13(13):

Epitranscriptomics: A New Layer of microRNA Regulation in Cancer.

Veronica De Paolis, Elisa Lorefice, Elisa Orecchini, Claudia Carissimi, Ilaria Laudadio, Valerio Fulci.

  MicroRNAs are pervasive regulators of gene expression at the post-transcriptional level in metazoan, playing key roles in several physiological and pathological processes. Accordingly, these small non-coding RNAs are also involved in cancer development and progression. Furthermore, miRNAs represent valuable diagnostic and prognostic biomarkers in malignancies. In the last twenty years, the role of RNA modifications in fine-tuning gene expressions at several levels has been unraveled. All RNA species may undergo post-transcriptional modifications, collectively referred to as epitranscriptomic modifications, which, in many instances, affect RNA molecule properties. miRNAs are not an exception, in this respect, and they have been shown to undergo several post-transcriptional modifications. In this review, we will summarize the recent findings concerning miRNA epitranscriptomic modifications, focusing on their potential role in cancer development and progression.

Keywords:  A-to-I editing; cancer; epitranscriptomics; m5C; m6A; m7G; microRNA

DOI:  https://doi.org/10.3390/cancers13133372
Angew Chem Int Ed Engl. 2021 Jul 21.

Single-cell Imaging of m6A modified RNA Using m6A-specific In Situ Hybridization mediated Proximity Ligation Assay (m6AISH-PLA).

Jinghong Li, Xiaojun Ren, Ruijie Deng, Kaixiang Zhang, Yupeng Sun, Yue Li.

  N 6 -methyladenosine (m 6 A) modification - the most prevalent mammalian RNA internal modification - plays key regulatory roles in mRNA metabolism. Current approaches for m 6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m 6 A-specific in situ hybridization mediated proximity ligation assay (m 6 AISH-PLA) for cellular imaging of m 6 A RNA, allowing to identify m 6 A modification at specific location in RNAs and image m 6 A RNA with single-cell and single-molecule resolution. Using m 6 AISH-PLA, we investigated the m 6 A level and subcellular location of HSP70 RNA103-m 6 A in each cell in response to heat shock, and found an increased m 6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock stress. m 6 AISH-PLA can serve in the study of m 6 A RNA in single-cell for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.

Keywords:  in situ imaging; m6A; proximity ligation; single-molecule; spatiotemporal information

DOI:  https://doi.org/10.1002/anie.202109118
Bioinformatics. 2021 Jul 22. pii: btab539. [Epub ahead of print]

ModPhred: an integrative toolkit for the analysis and storage of nanopore sequencing DNA and RNA modification data.

Leszek P Pryszcz, Eva Maria Novoa.

MOTIVATION: DNA and RNA modifications can now be identified using Nanopore sequencing. However, we currently lack a flexible software to efficiently encode, store, analyze and visualize DNA and RNA modification data.
RESULTS: Here we present ModPhred, a versatile toolkit that facilitates DNA and RNA modification analysis from nanopore sequencing reads in a user-friendly manner. ModPhred integrates probabilistic DNA and RNA modification information within the FASTQ and BAM file formats, can be used to encode multiple types of modifications simultaneously, and its output can be easily coupled to genomic track viewers, facilitating the visualization and analysis of DNA and RNA modification information in individual reads in a simple and computationally efficient manner.
AVAILABILITY: ModPhred is available at https://github.com/novoalab/modPhred, is implemented in Python3, and is released under an MIT license. Docker images with all dependencies preinstalled are also provided.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btab539
Neurochem Res. 2021 Jul 23.

ALKBH5 Promotes the Proliferation of Glioma Cells via Enhancing the mRNA Stability of G6PD.

Zhifeng Liu, Yonghan Chen, Leilei Wang, Suzhen Ji.

  This study aims to investigate the biological role of 6-methyladenine (m6A) methylation in inducing the carcinogenesis of glioma and its proliferation. Relative levels of ALKBH5 and glucose-6-phosphate dehydrogenase (G6PD) in glioma tissues and cell lines were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Gain-of-function and loss-of-function approaches were used to investigate the role of ALKBH5 in mediating proliferation and energy metabolism of glioma cells. The regulatory effect of ALKBH5 on G6PD was analyzed using m6A-qRT-PCR. Our results showed that ALKBH5 was upregulated in glioma, which stimulated glioma cells to proliferate. Serving as a m6A eraser, ALKBH5 demethylated the target transcript G6PD and enhanced its mRNA stability, thereby promoting G6PD translation and activating the pentose phosphate pathway (PPP). Collectively, ALKBH5 stimulates glioma cells to proliferate through erasing the m6A methylation of G6PD, which can be utilized as a potential therapeutic target for glioma.

Keywords:  ALKBH5; G6PD; Glioma; m6A modification

DOI:  https://doi.org/10.1007/s11064-021-03408-9
Front Plant Sci. 2021 ;12 701683

Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus.

Luis Alvarado-Marchena, Joan Marquez-Molins, Mireya Martinez-Perez, Frederic Aparicio, Vicente Pallás.

  N6-methyladenosine (m6A) modification is a dynamically regulated RNA modification that impacts many cellular processes and pathways. This epitranscriptomic methylation relies on the participation of RNA methyltransferases (referred to as "writers") and demethylases (referred to as "erasers"), respectively. We previously demonstrated that the Arabidopsis thaliana protein atALKBH9B showed m6A-demethylase activity and interacted with the coat protein (CP) of alfalfa mosaic virus (AMV), causing a profound impact on the viral infection cycle. To dissect the functional activity of atALKBH9B in AMV infection, we performed a protein-mapping analysis to identify the putative domains required for regulating this process. In this context, the mutational analysis of the protein revealed that the residues between 427 and 467 positions are critical for in vitro binding to the AMV RNA. The atALKBH9B amino acid sequence showed intrinsically disordered regions (IDRs) located at the N-terminal part delimiting the internal AlkB-like domain and at the C-terminal part. We identified an RNA binding domain containing an RGxxxRGG motif that overlaps with the C-terminal IDR. Moreover, bimolecular fluorescent experiments allowed us to determine that residues located between 387 and 427 are critical for the interaction with the AMV CP, which should be critical for modulating the viral infection process. Finally, we observed that atALKBH9B deletions of either N-terminal 20 residues or the C-terminal's last 40 amino acids impede their accumulation in siRNA bodies. The involvement of the regions responsible for RNA and viral CP binding and those required for its localization in stress granules in the viral cycle is discussed.

Keywords:  ALKBH; Ncpsdummy6-methyladenosine; RNA covalent modifications; RNA demethylases; RNA-binding proteins; alfamovirus; epitranscriptomics; plant viruses

DOI:  https://doi.org/10.3389/fpls.2021.701683