bims-rimeca 2021-06-06 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2021–06–06
25 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

Targeted RNA m6A Editing Using Engineered CRISPR-Cas9 Conjugates.
Comprehensive Analysis of PD-L1 Expression, Immune Infiltrates, and m6A RNA Methylation Regulators in Esophageal Squamous Cell Carcinoma.
METTL16 promotes cell proliferation by up-regulating cyclin D1 expression in gastric cancer.
The N6-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6.
YTHDF1 promotes hepatocellular carcinoma progression via activating PI3K/AKT/mTOR signaling pathway and inducing epithelial-mesenchymal transition.
EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing.
EDLm6APred: ensemble deep learning approach for mRNA m6A site prediction.
Novel prognostic implications of YTH domain family 2 in resected hepatocellular carcinoma.
The m6A methyltransferase METTL3 promotes the stemness and malignant progression of breast cancer by mediating m6A modification on SOX2.
Mapping of m6A and Its Regulatory Targets in Prostate Cancer Reveals a METTL3-low Induction of Therapy Resistance.
Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing.
Relative Quantification of Residue-Specific m6A RNA Methylation Using m6A-RT-QPCR.
Co-expression Network Revealed Roles of RNA m6A Methylation in Human β-Cell of Type 2 Diabetes Mellitus.
Human MettL3-MettL14 RNA adenine methyltransferase complex is active on double-stranded DNA containing lesions.
Potential Impact of ALKBH5 and YTHDF1 on Tumor Immunity in Colon Adenocarcinoma.
Rational Design of Novel Anticancer Small-Molecule RNA m6A Demethylase ALKBH5 Inhibitors.
PD-L1 and Immune Infiltration of m6A RNA Methylation Regulators and Its miRNA Regulators in Hepatocellular Carcinoma.
Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.
Programmable RNA N1-Methyladenosine Demethylation by a Cas13d-Directed Demethylase.
RNA Post-Transcriptional Modification Mapping Data Analysis Using RNA Framework.
Fat mass and obesity-associated protein (FTO) mediates signal transducer and activator of transcription 3 (STAT3)-drived resistance of breast cancer to doxorubicin.
Generation of a FTO gene knockout human embryonic stem cell line using CRISPR/Cas9 editing.
AlkAniline-Seq: A Highly Sensitive and Specific Method for Simultaneous Mapping of 7-Methyl-guanosine (m7G) and 3-Methyl-cytosine (m3C) in RNAs by High-Throughput Sequencing.
Monitoring the 5-Methoxycarbonylmethyl-2-Thiouridine (mcm5s2U) Modification Utilizing the Gamma-Toxin Endonuclease.
DNMT3A Mutation-Induced CDK1 Overexpression Promotes Leukemogenesis by Modulating the Interaction between EZH2 and DNMT3A.

Methods Mol Biol. 2021 ;2298 399-414

Targeted RNA m6A Editing Using Engineered CRISPR-Cas9 Conjugates.

Xiao-Min Liu, Shu-Bing Qian.

  N6-methyladenosine (m6A) is a major epitranscriptomic mark exerting crucial diverse roles in RNA metabolisms, including RNA stability, mRNA translation, and RNA structural rearrangement. m6A modifications at different RNA regions may have distinct molecular effects. Here, we describe a CRISPR-Cas9-based approach that enables targeted m6A addition or removal on endogenous RNA molecules without altering the nucleotide sequence. By fusing a catalytically inactive Cas9 with engineered m6A modification enzymes, the programmable m6A editors are capable of achieving RNA methylation and demethylation at desired sites, facilitating dissection of regional effects of m6A and diversifying the toolkits for RNA manipulation.

Keywords:  CRISPR-Cas9; Demethylation; Methylation; N6-methyladenosine; RNA targeting

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_23
Front Immunol. 2021 ;12 669750

Comprehensive Analysis of PD-L1 Expression, Immune Infiltrates, and m6A RNA Methylation Regulators in Esophageal Squamous Cell Carcinoma.

Wei Guo, Fengwei Tan, Qilin Huai, Zhen Wang, Fei Shao, Guochao Zhang, Zhenlin Yang, Renda Li, Qi Xue, Shugeng Gao, Jie He.

   Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common cancer types and represents a threat to global public health. N6-Methyladenosine (m6A) methylation plays a key role in the occurrence and development of many tumors, but there are still few studies investigating ESCC. This study attempts to construct a prognostic signature of ESCC based on m6A RNA methylation regulators and to explore the potential association of these regulators with the tumor immune microenvironment (TIME).
Methods: The transcriptome sequencing data and clinical information of 20 m6A RNA methylation regulators in 453 patients with ESCC (The Cancer Genome Atlas [TCGA] cohort, n = 95; Gene Expression Omnibus [GEO] cohort, n = 358) were obtained. The differing expression levels of m6A regulators between ESCC and normal tissue were evaluated. Based on the expression of these regulators, consensus clustering was performed to investigate different ESCC clusters. PD-L1 expression, immune score, immune cell infiltration and potential mechanisms among different clusters were examined. LASSO Cox regression analysis was utilized to obtain a prognostic signature based on m6A RNA methylation modulators. The relationship between the risk score based on the prognostic signature and the TIME of ESCC patients was studied in detail.
Results: Six m6A regulators (METTL3, WTAP, IGF2BP3, YTHDF1, HNRNPA2B1 and HNRNPC) were observed to be significantly highly expressed in ESCC tissues. Two molecular subtypes (clusters 1/2) were determined by consensus clustering of 20 m6A modulators. The expression level of PD-L1 in ESCC tissues increased significantly and was significantly negatively correlated with the expression levels of YTHDF2, METL14 and KIAA1429. The immune score, CD8 T cells, resting mast cells, and regulatory T cells (Tregs) in cluster 2 were significantly increased. Gene set enrichment analysis (GSEA) shows that this cluster involves multiple hallmark pathways. We constructed a five-gene prognostic signature based on m6A RNA methylation, and the risk score based on the prognostic signature was determined to be an independent prognostic indicator of ESCC. More importantly, the prognostic value of the prognostic signature was verified using another independent cohort. m6A regulators are related to TIME, and their copy-number alterations will dynamically affect the number of tumor-infiltrating immune cells.
Conclusion: Our study established a strong prognostic signature based on m6A RNA methylation regulators; this signature was able to accurately predict the prognosis of ESCC patients. The m6A methylation regulator may be a key mediator of PD-L1 expression and immune cell infiltration and may strongly affect the TIME of ESCC.

Keywords:  N6-methyladenosine methylation; PD-L1; RNA methylation; esophageal squamous cell carcinoma; immune infiltrates; prognosis

DOI:  https://doi.org/10.3389/fimmu.2021.669750
J Cell Mol Med. 2021 Jun 02.

METTL16 promotes cell proliferation by up-regulating cyclin D1 expression in gastric cancer.

Xiao-Kun Wang, Ya-Wei Zhang, Chun-Ming Wang, Bo Li, Tian-Zhi Zhang, Wen-Jie Zhou, Lyu-Jia Cheng, Ming-Yu Huo, Chang-Hua Zhang, Yu-Long He.

  N6-methyladenosine (m6A) is a well-known modification of RNA. However, as a key m6A methyltransferase, METTL16 has not been thoroughly studied in gastric cancer (GC). Here, the biological role of METTL16 in GC and its underlying mechanism was studied. Immunohistochemistry was used to detect the expression of METTL16 and relationship between METTL16 level and prognosis of GC was analysed. CCK8, colony formation assay, EdU assay and xenograft mouse model were used to study the effect of METTL16. Regulatory mechanism of METTL16 in the progression of GC was studied through flow cytometry analysis, RNA degradation assay, methyltransferase inhibition assay, RT-qPCR and Western blotting. METTL16 was highly expressed in GC cells and tissues and was associated with prognosis. In vitro and in vivo experiments confirmed that METTL16 promoted proliferation of GC cells and tumour growth. Furthermore, down-regulation of METTL16 inhibited proliferation by G1/S blocking. Significantly, we identified cyclin D1 as a downstream effector of METTL16. Knock-down METTL16 decreased the overall level of m6A and the stability of cyclin D1 mRNA in GC cells. Meanwhile, inhibition of methyltransferase activity reduced the level of cyclin D1. METTL16-mediated m6A methylation promotes proliferation of GC cells through enhancing cyclin D1 expression.

Keywords:  METTL16; cyclin D1; gastric cancer; m6A

DOI:  https://doi.org/10.1111/jcmm.16664
Cell Death Dis. 2021 Jun 04. 12(6): 578

The N6-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6.

Zhaofeng Li, Peng Wang, Jinteng Li, Zhongyu Xie, Shuizhong Cen, Ming Li, Wenjie Liu, Guiwen Ye, Guan Zheng, Mengjun Ma, Shan Wang, Wenhui Yu, Yanfeng Wu, Huiyong Shen.

N6-methyladenosine (m6A) modification is widespread in messenger RNAs and increasing evidence suggests the crucial roles of m6A in cell differentiation and tissue development. However, whether m6A modulates the osteogenic differentiation of mesenchymal stem cells (MSCs) has not been fully elucidated. Here we show that conditional knockout of the demethylase Alkbh5 in bone marrow MSCs strengthened bone mass in mice. Loss- and gain-of-function studies demonstrated that ALKBH5 negatively regulates the osteogenic differentiation of MSCs in vitro. At a mechanistic level, meRIP-seq and RNA-seq in MSCs following knockdown of ALKBH5 revealed changes in transcripts of PRMT6 containing consensus m6A motifs required for demethylation by ALKBH5. Furthermore, we found that ALKBH5 accelerates the degradation rate of PRMT6 mRNA in an m6A-dependent manner, and that the ALKBH5-PRMT6 axis regulates the osteogenesis of MSCs, mainly through activation of the PI3K/AKT pathway. Thus, our work reveals a different facet of the novel ALKBH5-PRMT6 axis that modulates the osteogenic differentiation of MSCs, which can serve as a target to improve the clinical use of MSCs.

DOI: https://doi.org/10.1038/s41419-021-03869-4
Exp Hematol Oncol. 2021 Jun 04. 10(1): 35

YTHDF1 promotes hepatocellular carcinoma progression via activating PI3K/AKT/mTOR signaling pathway and inducing epithelial-mesenchymal transition.

Xiangyuan Luo, Mengdie Cao, Fan Gao, Xingxing He.

   BACKGROUND: N6-methyladenosine (m6A) modification, as the most abundant RNA modification, widely participates in the physiological process and is involved in multiple disease progression, especially cancer. YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) is a pivotal m6A "reader" protein, which has been reported in multiple cancers. However, the role and molecular mechanism of YTHDF1 in HCC are still not fully elucidated.
METHODS: Based on various bioinformatics databases, q-RT PCR, western blot, and a tissue microarray containing 90 HCC samples, we examined the expression of YTHDF1 in HCC. Then, we applied the loss-of-function experiments to explore the role of YTHDF1 in HCC by in vitro and in vivo assays. Finally, we performed the gene set enrichment analysis (GSEA) to predict the potential signaling pathway of YTHDF1 involved in HCC and further verified this prediction.
RESULTS: YTHDF1 was overexpressed in HCC and associated with HCC grade. Depletion of YTHDF1 markedly impaired the proliferation, migration, invasion, and cell cycle process of HCC cells. Mechanistically, YTHDF1 promoted the growth of HCC cells via activating the PI3K/AKT/mTOR signaling pathway. Moreover, we also demonstrated that the epithelial-mesenchymal transition (EMT) mediated the promoting effect of YTHDF1 on the migration and invasion of HCC cells.
CONCLUSIONS: YTHDF1 contributes to the progression of HCC by activating PI3K/AKT/mTOR signaling pathway and inducing EMT.

Keywords:  EMT; Liver cancer; PI3K/AKT/mTOR; YTHDF1; m6A

DOI:  https://doi.org/10.1186/s40164-021-00227-0
Methods Mol Biol. 2021 ;2298 31-52

EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing.

Huanle Liu, Oguzhan Begik, Eva Maria Novoa.

  RNA modifications play pivotal roles in the RNA life cycle and RNA fate, and are now appreciated as a major posttranscriptional regulatory layer in the cell. In the last few years, direct RNA nanopore sequencing (dRNA-seq) has emerged as a promising technology that can provide single-molecule resolution maps of RNA modifications in their native RNA context. While native RNA can be successfully sequenced using this technology, the detection of RNA modifications is still challenging. Here, we provide an upgraded version of EpiNano (version 1.2), an algorithm to predict m6A RNA modifications from dRNA-seq datasets. The latest version of EpiNano contains models for predicting m6A RNA modifications in dRNA-seq data that has been base-called with Guppy. Moreover, it can now train models with features extracted from both base-called dRNA-seq FASTQ data and raw FAST5 nanopore outputs. Finally, we describe how EpiNano can be used in stand-alone mode to extract base-calling "error" features and current intensity information from dRNA-seq datasets. In this chapter, we provide step-by-step instructions on how to produce in vitro transcribed constructs to train EpiNano, as well as detailed information on how to use EpiNano to train, test, and predict m6A RNA modifications in dRNA-seq data.

Keywords:  Base-calling “errors”; Direct RNA sequencing; In vitro transcription; N6-methyladenosine; Nanopore sequencing; Native RNA; Oxford Nanopore Technologies; RNA modification; Support vector machine

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_3
BMC Bioinformatics. 2021 May 29. 22(1): 288

EDLm6APred: ensemble deep learning approach for mRNA m6A site prediction.

Lin Zhang, Gangshen Li, Xiuyu Li, Honglei Wang, Shutao Chen, Hui Liu.

   BACKGROUND: As a common and abundant RNA methylation modification, N6-methyladenosine (m6A) is widely spread in various species' transcriptomes, and it is closely related to the occurrence and development of various life processes and diseases. Thus, accurate identification of m6A methylation sites has become a hot topic. Most biological methods rely on high-throughput sequencing technology, which places great demands on the sequencing library preparation and data analysis. Thus, various machine learning methods have been proposed to extract various types of features based on sequences, then occupied conventional classifiers, such as SVM, RF, etc., for m6A methylation site identification. However, the identification performance relies heavily on the extracted features, which still need to be improved.
RESULTS: This paper mainly studies feature extraction and classification of m6A methylation sites in a natural language processing way, which manages to organically integrate the feature extraction and classification simultaneously, with consideration of upstream and downstream information of m6A sites. One-hot, RNA word embedding, and Word2vec are adopted to depict sites from the perspectives of the base as well as its upstream and downstream sequence. The BiLSTM model, a well-known sequence model, was then constructed to discriminate the sequences with potential m6A sites. Since the above-mentioned three feature extraction methods focus on different perspectives of m6A sites, an ensemble deep learning predictor (EDLm6APred) was finally constructed for m6A site prediction. Experimental results on human and mouse data sets show that EDLm6APred outperforms the other single ones, indicating that base, upstream, and downstream information are all essential for m6A site detection. Compared with the existing m6A methylation site prediction models without genomic features, EDLm6APred obtains 86.6% of the area under receiver operating curve on the human data sets, indicating the effectiveness of sequential modeling on RNA. To maximize user convenience, a webserver was developed as an implementation of EDLm6APred and made publicly available at www.xjtlu.edu.cn/biologicalsciences/EDLm6APred .
CONCLUSIONS: Our proposed EDLm6APred method is a reliable predictor for m6A methylation sites.

Keywords:  Deep learning; Predictor; Word embedding; m6A methylation modification

DOI:  https://doi.org/10.1186/s12859-021-04206-4
Oncol Lett. 2021 Jul;22(1): 538

Novel prognostic implications of YTH domain family 2 in resected hepatocellular carcinoma.

Nobuhiko Nakagawa, Fuminori Sonohara, Katsuhito Tanaka, Yuki Sunagawa, Yoshikuni Inokawa, Hideki Takami, Masamichi Hayashi, Suguru Yamada, Mitsuro Kanda, Chie Tanaka, Goro Nakayama, Masahiko Koike, Yasuhiro Kodera.

  N6-methyladenosine (m6A), the most abundant internal RNA modification, serves a critical role in cancer development. However, the clinical implications of m6A in hepatocellular carcinoma (HCC) remain unclear. The present study sought to reveal the potential roles of m6A readers, which recognize m6A, in HCC. A total of 177 HCC and paired non-cancerous liver tissues from patients who underwent hepatectomy were analysed using quantitative PCR for the expression of m6A readers: YT521-B homology domain family 1 (YTHDF1) and YT521-B homology domain family 2 (YTHDF2). The expression levels of both YTHDF1 and YTHDF2 were not significantly different between tumour and non-cancerous tissues (P=0.93 and P=0.7, respectively). Analysis of the association between clinical features and m6A reader expression revealed that YTHDF1 expression was associated with formation of capsule (P=0.02), whereas low YTHDF2 expression was associated with septal formation (P=0.02). Furthermore, high YTHDF1 expression and high YTHDF2 expression were significantly associated with shorter recurrence-free survival (RFS) [YTHDF1: Mean survival time (MST), 34.0 vs. 19.0 months, P=0.014; YTHDF2: MST, 30.1 vs. 12.9 months, P=0.0032], whereas YTHDF1 and YTHDF2 expression was not significantly associated with overall survival (OS) (YTHDF1: MST, 99.4 vs. 70.2 months, P=0.74; YTHDF2: MST, 98.4 vs. 64.1 months, P=0.28). According to multivariate analysis, serosal invasion [hazard ratio (HR), 2.39; 95% CI 1.30-4.42; P=0.005), portal vein or hepatic vein invasion (HR, 2.82; 95% CI 1.26-6.28; P=0.01) and YTHDF2 expression in HCC tissues (HR, 1.85; 95% CI 1.09-3.15; P=0.02) were identified as significant independent prognostic factors for RFS. α-fetoprotein (HR, 1.79; 95% CI 1.10-2.92; P=0.02), serosal invasion (HR, 1.99; 95% CI 1.17-3.34; P=0.01) and portal vein or hepatic vein invasion (HR, 3.02; 95% CI 1.38-6.61; P=0.006) were identified as significant independent prognostic factors for OS. In conclusion, the present study revealed that high YTHDF2 expression, an m6A reader, in HCC tissues was associated with cancer recurrence.

Keywords:  YTH domain family; hepatocellular carcinoma; methylation of N6 adenosine; prognosis

DOI:  https://doi.org/10.3892/ol.2021.12799
J BUON. 2021 Mar-Apr;26(2):26(2): 444-449

The m6A methyltransferase METTL3 promotes the stemness and malignant progression of breast cancer by mediating m6A modification on SOX2.

Jiping Xie, Jinling Ba, Min Zhang, Yi Wan, Zeyu Jin, Yonqiang Yao.

PURPOSE: We aimed to uncover the role of METTL3 in stimulating the stemness and progression of breast cancer (BCa) through mediating N6-methyladenosine (m6A) modification on SOX2 mRNA.
METHODS: METTL3 levels in 48 paired BCa and adjacent normal ones were examined. Kaplan-Meier method was introduced for assessing the prognostic value of METTL3 in BCa. Regulatory effects of METTL3 on invasive and migratory abilities in MCF-7 cells were evaluated by Transwell assay. Besides, the protein levels of SOX2 and tumor stem cell markers CD133 and CD44 in MCF-7 cells affected by METTL3 were determined by Western blot. In addition, the potential interaction between METTL3 and SOX2 was ascertained through RIP (RNA-Binding Protein Immunoprecipitation) assay. Moreover, the interaction between IGF2BP2 and SOX2 influenced by METTL3 was verified by RIP assay as well.
RESULTS: METTL3 was upregulated in BCa tissues, especially in T3-T4 or those accompanied with lymphatic metastasis. BCa patients expressing a high level of METTL3 suffered worse prognosis. Knockdown of METTL3 downregulated protein levels of SOX2, CD133 and CD44 in MCF-7 cells. Moreover, invasive and migratory abilities were attenuated in BCa cells with METTL3 knockdown. Silencing of IGF2BP2 markedly downregulated SOX2. RIP assay confirmed the binding between METTL3 and SOX2 mRNA, and knockdown of METTL3 decreased the enrichment of SOX2 in anti-IGF2BP2. Interestingly, overexpression of SOX2 partially reversed the regulatory effects of downregulated METTL3 on MCF-7 cells.
CONCLUSIONS: METTL3 is upregulated in BCa, and it promotes the stemness and malignant progression of BCa through mediating m6A modification on SOX2 mRNA.
Mol Cancer Res. 2021 Jun 04. pii: molcanres.MCR-21-0014-E.2021. [Epub ahead of print]

Mapping of m6A and Its Regulatory Targets in Prostate Cancer Reveals a METTL3-low Induction of Therapy Resistance.

Kellie A Cotter, John Gallon, Nadine Uebersax, Philip Rubin, Kate D Meyer, Salvatore Piscuoglio, Samie R Jaffrey, Mark A Rubin.

Recent evidence has highlighted the role of N6-methyladenosine (m6A) in the regulation of mRNA expression, stability and translation, supporting a potential role for post-transcriptional regulation mediated by m6A in cancer. Here we explore prostate cancer as an exemplar and demonstrate that low levels of N6-adenosine-methyltransferase (METTL3) is associated with advanced metastatic disease. To investigate this relationship, we generated the first prostate m6A maps, and further examined how METTL3 regulates expression at the level of transcription, translation, and protein. Significantly, transcripts encoding extracellular matrix proteins are consistently upregulated with METTL3 knockdown. We also examined the relationship between METTL3 and androgen signaling and discovered the upregulation of a hepatocyte nuclear factor-driven gene signature that is associated with therapy resistance in prostate cancer. Significantly, METTL3 knockdown rendered the cells resistant to androgen receptor antagonists via an androgen receptor independent mechanism driven by the upregulation of nuclear receptor NR5A2/LRH-1. Implications: These findings implicate changes in m6A as a mechanism for therapy resistance in metastatic prostate cancer.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0014
Methods Mol Biol. 2021 ;2298 123-134

Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing.

Bryan R Cullen, Kevin Tsai.

  Epitranscriptomic RNA modifications function as an important layer of gene regulation that modulates the function of RNA transcripts. A key step in understanding how RNA modifications regulate biological processes is the mapping of their locations, which is most commonly done by RNA immunoprecipitation (RIP) using modification-specific antibodies. Here, we describe the use of a photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) method, in conjunction with RNA modification-specific antibodies, to map modification sites. First described as photo-crosslinking-assisted m6A sequencing (PA-m6A-seq), this method allows the mapping of RNA modifications at a higher resolution, with lower background than traditional RIP, and can be adapted to any RNA modification for which a specific antibody is available.

Keywords:  Epitranscriptomic RNA modification; RNA immunoprecipitation; ac4C; m5C; m6A

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_8
Methods Mol Biol. 2021 ;2298 185-195

Relative Quantification of Residue-Specific m6A RNA Methylation Using m6A-RT-QPCR.

Ane Olazagoitia-Garmendia, Ainara Castellanos-Rubio.

  Technological advances in high-throughput sequencing in combination with antibody enrichment and/or induced nucleotide-specific chemical modifications have accelerated the mapping of epitranscriptomic modifications. However, site-specific detection and quantification of m6A are still technically challenging. Here, we describe a simple RT-QPCR-based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues.

Keywords:  QPCR; RNA methylation; Retrotranscription; m6A

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_12
Front Cell Dev Biol. 2021 ;9 651142

Co-expression Network Revealed Roles of RNA m6A Methylation in Human β-Cell of Type 2 Diabetes Mellitus.

Cong Chen, Qing Xiang, Weilin Liu, Shengxiang Liang, Minguang Yang, Jing Tao.

  RNA m6A methylation plays an important role in the pathogenesis of type 2 diabetes mellitus (T2DM). RNA modifications and RNA-modifying regulators have recently emerged as critical factors involved in β-cell function and insulin resistance, including "writers," "erasers," and "readers." However, their key roles in regulating gene expression in T2DM remain unclear. The construction of co-expression network could provide a cue to resolve this complex regulatory pathway. We collected the transcriptome datasets of β-cell in diabetic patients, calculated the partial correlation coefficient, excluded the influence from control variables of diabetes related genes, and identified the genes significantly co-expressed with m6A regulators. A total of 985 genes co-expressed with m6A regulators (Co-m6AR) were identified, which were enriched in metabolic process, MAPK and EGFR signaling pathways. Some of them have been confirmed to play a pivotal role in T2DM, including CCNL2, CSAD, COX5A, GAB2, and MIRLET7I, etc. Further, we analyzed the m6A modification characteristics of Co-m6AR in β-cell and identified 228 Co-m6AR containing m6A methylation sites, involving in several key signaling pathways regulating T2DM. We finally screened out 13 eQTL-SNPs localized in Co-m6ARs, and 4 have been reported strongly associated with diabetes, including GAB2, LMNB2, XAB2, and RBM39. This co-expression analysis provides important information to reveal the potential regulatory mechanism of RNA m6A methylation in T2DM.

Keywords:  RNA m6A methylation; RNA m6A methyltransferase; co-expression network; insulin; type 2 diabetes mellitus

DOI:  https://doi.org/10.3389/fcell.2021.651142
Nucleic Acids Res. 2021 Jun 04. pii: gkab460. [Epub ahead of print]

Human MettL3-MettL14 RNA adenine methyltransferase complex is active on double-stranded DNA containing lesions.

Dan Yu, John R Horton, Jie Yang, Taraneh Hajian, Masoud Vedadi, Cari A Sagum, Mark T Bedford, Robert M Blumenthal, Xing Zhang, Xiaodong Cheng.

MettL3-MettL14 methyltransferase complex has been studied widely for its role in RNA adenine methylation. This complex is also recruited to UV- and X-ray exposed DNA damaged sites, and its methyltransfer activity is required for subsequent DNA repair, though in theory this could result from RNA methylation of short transcripts made at the site of damage. We report here that MettL3-MettL14 is active in vitro on double-stranded DNA containing a cyclopyrimidine dimer - a major lesion of UV radiation-induced products - or an abasic site or mismatches. Furthermore, N6-methyladenine (N6mA) decreases misincorporation of 8-oxo-guanine (8-oxoG) opposite to N6mA by repair DNA polymerases. When 8-oxoG is nevertheless incorporated opposite N6mA, the methylation inhibits N6mA excision from the template (correct) strand by the adenine DNA glycosylase (MYH), implying that the methylation decreases inappropriate misrepair. Finally, we observed that the N6mA reader domain of YTHDC1, which is also recruited to sites of DNA damage, binds N6mA that is located across from a single-base gap between two canonical DNA helices. This YTHDC1 complex with a gapped duplex is structurally similar to DNA complexes with FEN1 and GEN1 - two members of the nuclease family that act in nucleotide excision repair, mismatch repair and homologous recombination, and which incise distinct non-B DNA structures. Together, the parts of our study provide a plausible mechanism for N6mA writer and reader proteins acting directly on lesion-containing DNA, and suggest in vivo experiments to test the mechanisms involving methylation of adenine.

DOI: https://doi.org/10.1093/nar/gkab460
Front Oncol. 2021 ;11 670490

Potential Impact of ALKBH5 and YTHDF1 on Tumor Immunity in Colon Adenocarcinoma.

Guanyu Yan, Yue An, Boyang Xu, Ningning Wang, Xuren Sun, Mingjun Sun.

   Background: ALKBH5 and YTHDF1 are regarded as the eraser and reader, respectively, in N6-methyladenosine (m6A) modification. Recently, immune contexture has been drawing increasing attention in terms of the progression and treatment of cancers. This study aimed to determine the relationship between ALKBH5/YTHDF1 and immunological characteristics of colon adenocarcinoma (COAD).
Methods: Expression of ALKBH5 and YTHDF1 was investigated across TCGA and GEO validated in our study. Patients with COAD were divided into two clusters using consensus clustering based on the expression of ALKBH5 and YTHDF1. We then compared their clinical characteristics and performed gene set enrichment analysis (GSEA) to identify the functional differences. Immune infiltration analyses were conducted using ESTIMATE, CIBERSORT, and ssGSEA. In addition, we evaluated the expression of the targets of immune checkpoint inhibitors (ICIs) and calculated the tumor mutation burden (TMB) of the tumor samples. Weighted gene co-expression network analysis (WGCNA) was used to identify the genes related to both ALKBH5/YTHDF1 expression and immunity. GSE39582 was utilized for external validation of immunological features between the two clusters.
Results: Cluster 2 had high expression of ALKBH5 and lesser so of YTHDF1, whereas Cluster 1 had just the reverse. Cluster 1 had a higher N stage and pathological stage than Cluster 2. The latter had stronger immune infiltration, higher expression of targets of ICIs, more TMB, and a larger proportion of deficiency in mismatch repair-microsatellite instability-high (dMMR-MSI-H) status than Cluster 1. Moreover, WGCNA revealed 14 genes, including PD1 and LAG3, related to both the expression of ALKBH5/YTHDF1 and immune scores.
Conclusions: ALKBH5 and YTHDF1 influence immune contexture and can potentially transform cold tumors into hot tumors in patients with COAD.

Keywords:  ALKBH5; YTHDF1; colon adenocarcinoma; immune contexture; m6A modification

DOI:  https://doi.org/10.3389/fonc.2021.670490
ACS Omega. 2021 May 25. 6(20): 13310-13320

Rational Design of Novel Anticancer Small-Molecule RNA m6A Demethylase ALKBH5 Inhibitors.

Simona Selberg, Neinar Seli, Esko Kankuri, Mati Karelson.

The RNA 6-N-methyladenosine (m6A) demethylase ALKBH5 has been shown to be oncogenic in several cancer types, including leukemia and glioblastoma. We present here the target-tailored development and first evaluation of the antiproliferative effects of new ALKBH5 inhibitors. Two compounds, 2-[(1-hydroxy-2-oxo-2-phenylethyl)sulfanyl]acetic acid (3) and 4-{[(furan-2-yl)methyl]amino}-1,2-diazinane-3,6-dione (6), with IC50 values of 0.84 μM and 1.79 μM, respectively, were identified in high-throughput virtual screening of the library of 144 000 preselected compounds and subsequent verification of hits in an m6A antibody-based enzyme-linked immunosorbent assay (ELISA) enzyme inhibition assay. The effect of these compounds on the proliferation of selected target cancer cell lines was then measured. In the case of three leukemia cell lines (HL-60, CCRF-CEM, and K562) the cell proliferation was suppressed at low micromolar concentrations of inhibitors, with IC50 ranging from 1.38 to 16.5 μM. However, the effect was low or negligible in the case of another leukemia cell line, Jurkat, and the glioblastoma cell line A-172. These results demonstrate the potential of ALKBH5 inhibition as a cancer-cell-type-selective antiproliferative strategy.

DOI: https://doi.org/10.1021/acsomega.1c01289
Biomed Res Int. 2021 ;2021 5516100

PD-L1 and Immune Infiltration of m6A RNA Methylation Regulators and Its miRNA Regulators in Hepatocellular Carcinoma.

Yingxue Lin, Yinhui Yao, Ying Wang, Lingdi Wang, Haipeng Cui.

Background: The aim of this study was to systematically evaluate the relationship between the expression of m6A RNA methylation regulators and prognosis in HCC.
Methods: We compared the expression of m6A methylation modulators and PD-L1 between HCC and normal in TCGA database. HCC samples were divided into two subtypes by consensus clustering of data from m6A RNA methylation regulators. The differences in PD-L1, immune infiltration, and prognosis between the two subtypes were further compared. The LASSO regression was used to build a risk score for m6A modulators. In addition, we identified miRNAs that regulate m6A regulators.
Results: We found that fourteen m6A regulatory genes were significantly differentially expressed between HCC and normal. HCC samples were divided into two clusters. Of these, there are higher PD-L1 expression and poorer overall survival (OS) in cluster 1. There was a significant difference in immune cell infiltration between cluster 1 and cluster 2. Through the LASSO model, we obtained 12 m6A methylation regulators to construct a prognostic risk score. Compared with patients with a high-risk score, patients with a low-risk score had upregulated PD-L1 expression and worse prognosis. There was a significant correlation between risk score and tumor-infiltrating immune cells. Finally, we found that miR-142 may be the important regulator for m6A RNA methylation in HCC.
Conclusion: Our results suggest that m6A RNA methylation modulators may affect the prognosis through PD-L1 and immune cell infiltration in HCC patients. In addition, the two clusters may be beneficial for prognostic stratification and improving immunotherapeutic efficacy.

DOI: https://doi.org/10.1155/2021/5516100
Methods Mol Biol. 2021 ;2298 171-184

Assessing 2'-O-Methylation of mRNA Using Quantitative PCR.

Brittany A Elliott, Christopher L Holley.

  2'-O-methylation (Nm) is an RNA modification commonly found on rRNA and snRNA, and at the mRNA 5'-cap, but has more recently been found internally on mRNA. The study of internal Nm modifications on mRNA is in the early stages, but we have reported that this sort of Nm modification can regulate mRNA abundance and translation. Although there are many methods to determine the presence of Nm on rRNA, detecting Nm on specific mRNA transcripts is technically difficult because they are much less abundant than rRNA. Some of these methods rely on the fact that Nm modification of RNA disrupts reverse transcription reactions when performed at low dNTP concentrations. In this chapter, we describe our approach to using quantitative PCR in conjunction with reverse transcription at low dNTPs, which is sensitive enough to detect changes to Nm modification of mRNA.

Keywords:  2′-O-methylation; Low dNTP; RNA modifications; Reverse transcription; qPCR

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_11
Angew Chem Int Ed Engl. 2021 Jun 03.

Programmable RNA N1-Methyladenosine Demethylation by a Cas13d-Directed Demethylase.

Shanshan Xie, Hao Jin, Feng Yang, Hong Zheng, Yongxia Chang, Ying Liao, Ye Zhang, Tianhua Zhou, Yang Li.

  N 1 -methyladenosine (m 1 A) is a prevalent and reversible RNA modification, which plays a crucial role in the regulation of RNA fate and gene expression. However, the lack of tools to precisely manipulate m 1 A sites in specific transcripts has hindered efforts to clarify the association between a specific m 1 A-modified transcript and its phenotypic outcomes. Here we develop a CRISPR-Cas13d-based tool called re engineered m 1 A mo dification v alid er aser (termed "REMOVER") for targeted m 1 A demethylation of a specific transcript. The catalytically inactive RfxCas13d (dCasRx) is fused to the m 1 A demethylase ALKBH3, and the dCasRx-ALKBH3 fusion protein can mediate potent demethylation of m 1 A-modified RNAs. We further find that REMOVER can specifically demethylate m 1 A of MALAT1 and PRUNE1 RNAs, thereby significantly increasing their stability. Our study establishes REMOVER as a tool for targeted RNA demethylation of specific m 1 A-modified transcripts, which enables further elucidation of the relationship between m 1 A modification of specific transcripts and their phenotypic outcomes.

Keywords:  CRISPR-Cas13; N1-methyladenosine; RNA; mRNA; targeted demethylation

DOI:  https://doi.org/10.1002/anie.202105253
Methods Mol Biol. 2021 ;2298 3-13

RNA Post-Transcriptional Modification Mapping Data Analysis Using RNA Framework.

Ilaria Manfredonia, Danny Incarnato.

  RNA post-transcriptional modifications (PTMs) are progressively gaining relevance in the study of coding-independent functions of RNA. RNA PTMs act as dynamic regulators of several aspects of the RNA physiology, from translation to half-life. Rising interest is supported by the advance of high-throughput techniques enabling the detection of these modifications on a transcriptome-wide scale. To this end, here we illustrate the usefulness of RNA Framework, a comprehensive toolkit for the analysis of RNA PTM mapping experiments, by reanalyzing two published transcriptome-scale datasets of N1-methyladenosine (m1A) and pseudouridine (Ψ) mapping, based on two different experimental strategies.

Keywords:  High-throughput sequencing; N1-methyladenosine; Pseudouridine; RNA immunoprecipitation; RNA post-transcriptional modifications; m1A

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_1
Bioengineered. 2021 Dec;12(1): 1874-1889

Fat mass and obesity-associated protein (FTO) mediates signal transducer and activator of transcription 3 (STAT3)-drived resistance of breast cancer to doxorubicin.

Yan Wang, Zhiqiang Cheng, Jing Xu, Meina Lai, Liming Liu, Min Zuo, Lin Dang.

  Excessive activation of signal transducer and activator of transcription 3 (STAT3) is implicated in breast cancer (BC) chemoresistance, but its underlying mechanism is not fully understood. There are STAT3 binding sites in fat mass and obesity-associated protein (FTO) promoter region, thus STAT3 may regulate the transcription of FTO. This study aimed to investigate the correlation between FTO and STAT3 in BC chemoresistance. Herein, FTO and STAT3 were highly expressed in doxorubicin-resistant BC (BC-DoxR) cells. CHIP assay verified the binding between STAT3 and FTO promoter in BC-DoxR cells. Dual luciferase reporter assay showed that FTO promoter activity was inhibited by S3I-201 (STAT3 inhibitor) but enhanced by epidermal growth factor (EGF, STAT3 activator) in BC-DoxR and BC cells. FTO mRNA and protein expression were suppressed by S3I-201 in BC-DoxR cells and EGF-stimulated BC cells. Notably, FTO regulated total N6-methyladenosine (m6A) levels in BC-DoxR and BC cells but could not affect STAT3 mRNA expression, indicating that FTO was not involved in the m6A modification of STAT3. However, FTO could activate STAT3 signaling in BC-DoxR and BC cells. Besides, FTO knockdown inhibited the doxorubicin resistance of BC-DoxR cells, while FTO overexpression enhanced the doxorubicin resistance and weakened the doxorubicin sensitivity of BC cells. Moreover, decreased doxorubicin resistance by STAT3 knockdown was abolished by FTO overexpression and decreased doxorubicin sensitivity by STAT3 overexpression was reversed by FTO knockdown, indicating that FTO was implicated in STAT3-mediated doxorubicin resistance and impairment of doxorubicin sensitivity of BC cells. Altogether, our findings provide a mechanism underlying BC doxorubicin resistance.

Keywords:  Chemosensitivity; Doxorubicin resistance; FTO; Stat3; breast cancer

DOI:  https://doi.org/10.1080/21655979.2021.1924544
Stem Cell Res. 2021 May;pii: S1873-5061(21)00208-7. [Epub ahead of print]53 102362

Generation of a FTO gene knockout human embryonic stem cell line using CRISPR/Cas9 editing.

Cong Wei, Qian Luo, Binsheng Wang, Yan Long, Meng Zhang, Wei Shan, Xiaohong Yu, Yulin Xu, Pengxu Qian, He Huang.

Fat mass and obesity-associated protein (FTO) is the first protein found to have the activity of N6-methyladenosine (m6A) demethylation. It has been reported that FTO was involved in different physiological and pathological processes, including stem cell differentiation, sex determination, tumorigenesis, and progression. To further understand the exact role of FTO in these processes, we generated a FTO knockout human embryonic stem cell (hESC) line by CRISPR/Cas9 mediated gene editing method. This cell line maintained normal karyotype, pluripotency, and trilineage differentiation potential, which are considered as a model for function studies of the FTO protein in hESC self-renewal and differentiation.

DOI: https://doi.org/10.1016/j.scr.2021.102362
Methods Mol Biol. 2021 ;2298 77-95

AlkAniline-Seq: A Highly Sensitive and Specific Method for Simultaneous Mapping of 7-Methyl-guanosine (m7G) and 3-Methyl-cytosine (m3C) in RNAs by High-Throughput Sequencing.

Virginie Marchand, Lilia Ayadi, Valérie Bourguignon-Igel, Mark Helm, Yuri Motorin.

  Epitranscriptomics is an emerging field where the development of high-throughput analytical technologies is essential to profile the dynamics of RNA modifications under different conditions. Despite important advances during the last 10 years, the number of RNA modifications detectable by next-generation sequencing is restricted to a very limited subset. Here, we describe a highly efficient and fast method called AlkAniline-Seq to map simultaneously two different RNA modifications: 7-methyl-guanosine (m7G) and 3-methyl-cytosine (m3C) in RNA. Our protocol is based on three subsequent chemical/enzymatic steps allowing the enrichment of RNA fragments ending at position n + 1 to the modified nucleotide, without any prior RNA selection. Therefore, AlkAniline-Seq demonstrates an outstanding sensitivity and specificity for these two RNA modifications. We have validated AlkAniline-Seq using bacterial, yeast, and human total RNA, and here we present, as an example, a synthetic view of the complete profiling of these RNA modifications in S. cerevisiae tRNAs.

Keywords:  3-Methyl-cytosine; 7-Methyl-guanosine; Bacteria; High-throughput sequencing; RNA modification mapping

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_5
Methods Mol Biol. 2021 ;2298 197-216

Monitoring the 5-Methoxycarbonylmethyl-2-Thiouridine (mcm5s2U) Modification Utilizing the Gamma-Toxin Endonuclease.

Jenna M Lentini, Dragony Fu.

  The post-transcriptional modification of tRNAs at the wobble position plays a critical role in proper mRNA decoding and efficient protein synthesis. In particular, certain wobble uridines in eukaryotes are converted to 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The mcm5s2U modification modulates decoding during translation by increasing the stringency of the wobble uridine to base pair with its canonical nucleotide partner, thereby restricting decoding to its cognate codon. Here, we outline a technique to monitor wobble uridine status in mcm5s2U-containing tRNAs using the gamma-toxin endonuclease from the yeast Kluyveromyces lactis that naturally cleaves tRNAs containing the mcm5s2U modification. This technique is coupled to Northern blotting or reverse transcription-PCR to enable rapid and sensitive detection of changes in mcm5s2U modification state.

Keywords:  Endonuclease; Northern blotting; RNA modification; Wobble position; mcm5s2U; tRNA

DOI:  https://doi.org/10.1007/978-1-0716-1374-0_13
Biomolecules. 2021 May 22. pii: 781. [Epub ahead of print]11(6):

DNMT3A Mutation-Induced CDK1 Overexpression Promotes Leukemogenesis by Modulating the Interaction between EZH2 and DNMT3A.

Ying Yang, Yujun Dai, Xuejiao Yang, Songfang Wu, Yueying Wang.

  DNMT3A mutations are frequently identified in acute myeloid leukemia (AML) and indicate poor prognosis. Previously, we found that the hotspot mutation DNMT3A R882H could upregulate CDK1 and induce AML in conditional knock-in mice. However, the mechanism by which CDK1 is involved in leukemogenesis of DNMT3A mutation-related AML, and whether CDK1 could be a therapeutic target, remains unclear. In this study, using fluorescence resonance energy transfer and immunoprecipitation analysis, we discovered that increased CDK1 could compete with EZH2 to bind to the PHD-like motif of DNMT3A, which may disturb the protein interaction between EZH2 and DNMT3A. Knockdown of CDK1 in OCI-AML3 cells with DNMT3A mutation markedly inhibited proliferation and induced apoptosis. CDK1 selective inhibitor CGP74514A (CGP) and the pan-CDK inhibitor flavopiridol (FLA) arrested OCI-AML3 cells in the G2/M phase, and induced cell apoptosis. CGP significantly increased CD163-positive cells. Moreover, the combined application of CDK1 inhibitor and traditional chemotherapy drugs synergistically inhibited proliferation and induced apoptosis of OCI-AML3 cells. In conclusion, this study highlights CDK1 overexpression as a pathogenic factor and a potential therapeutic target for DNMT3A mutation-related AML.

Keywords:  CDK1; DNA methyltransferase 3A; acute myeloid leukemia; mutation; targeted therapy

DOI:  https://doi.org/10.3390/biom11060781