Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.     

Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N⁶-methyladenosine (m⁶A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m⁶A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m⁶A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.     

m6A甲基转移酶家族Fiona/METT-10/METTL16的功能

RNA碱基上的化学修饰在其功能的精准调节中发挥关键作用,其中m⁶A是自然界中最普遍的RNA修饰之一,且该修饰在调控RNA稳定性、pre-mRNA剪接、翻译等方面具有重要功能。在真核生物中,m⁶A修饰主要由两种甲基转移酶完成,其在哺乳动物中分别命名为METTL3和METTL16。与METTL3相似,METTL16的底物多种多样,包括pre-mRNA、rRNA、snRNA和lncRNA等,因此似乎难以用一种分子机理解释METTL16对不同RNA底物进行m⁶A修饰的功能。此外,METTL16还在翻译调控中发挥重要作用,但此过程不依赖其甲基转移酶活性,这进一步增加了高度保守的METTL16的功能复杂性。本综述总结了METTL16及其同源蛋白质的结构域、甲基化底物以及它们的潜在功能,着重阐述了在不同物种中关于METTL16研究结果的矛盾之处,并推测METTL16调控S-腺苷基甲硫氨酸(SAM)代谢的功能是趋同进化的一个潜在案例。     

Fat mass and obesity-associated protein regulates lipogenesis via m6A modification in fatty acid synthase mRNA

As the first identified N⁶-methyladenosine (m⁶A) demethylase, fat mass and obesity-associated (FTO) protein is associated with fatty acid synthase (FASN) and lipid accumulation. However, little is known about the regulatory role of FTO in the expression of FASN and de novo lipogenesis through m⁶A modification. In this study, we used FTO small interfering RNA to explore the effects of FTO knockdown on hepatic lipogenesis and its underlying epigenetic mechanism in HepG2 cells. We found that knockdown of FTO increased m⁶A levels in total RNA and enhanced the expression of YTH domain family member 2 which serves as the m⁶A-binding protein. The de novo lipogenic enzymes and intracellular lipid content were significantly decreased under FTO knockdown. Mechanistically, knockdown of FTO dramatically enhanced m⁶A levels in FASN messenger RNA (mRNA), leading to the reduced expression of FASN mRNA through m⁶A-mediated mRNA decay. The protein expressions of FASN along with acetyl CoA carboxylase and ATP-citrate lyase were further decreased, which inhibited de novo lipogenesis, thereby resulting in the deficiency of lipid accumulation in HepG2 cells and the induction of cellular apoptosis. The results reveal that FTO regulates hepatic lipogenesis via FTO-dependent m⁶A demethylation in FASN mRNA and indicate the critical role of FTO-mediated lipid metabolism in the survival of HepG2 cells. This study provides novel insights into a unique RNA epigenetic mechanism by which FTO mediates hepatic lipid accumulation through m⁶A modification and indicates that FTO could be a potential target for obesity-related diseases and cancer.     

METTL3-mediated mRNA N6-methyladenosine is required for oocyte and follicle development in mice

Proper follicle development is very important for the production of mature oocytes, which is essential for the maintenance of female fertility. This complex biological process requires precise gene regulation. The most abundant modification of mRNA, N⁶-methyladenosine (m⁶A), is involved in many RNA metabolism processes, including RNA splicing, translation, stability, and degradation. Here, we report that m⁶A plays essential roles during oocyte and follicle development. Oocyte-specific inactivation of the key m⁶A methyltransferase Mettl3 with Gdf9-Cre caused DNA damage accumulation in oocytes, defective follicle development, and abnormal ovulation. Mechanistically, combined RNA-seq and m⁶A methylated RNA immunoprecipitation sequencing (MeRIP-seq) data from oocytes revealed, that we found METTL3 targets Itsn2 for m⁶A modification and then enhances its stability to influence the oocytes meiosis. Taken together, our findings highlight the crucial roles of mRNA m⁶A modification in follicle development and coordination of RNA stabilization during oocyte growth.Subject terms: Oogenesis, Infertility     

p53 m6A modulation sensitizes hepatocellular carcinoma to apatinib through apoptosis

Hepatocellular carcinoma (HCC) is insidious and prone to metastasis and recurrence. Currently, no effective treatment is available for HCC. Furthermore, HCC does not respond to various radio- and chemotherapies, and the molecular mechanism of treatment resistance is unclear. Here, we found that p53 n6-methyladenosine (m⁶A) played a decisive role in regulating HCC sensitivity to chemotherapy via the p53 activator RG7112 and the vascular endothelial growth factor receptor inhibitor apatinib. Our results reveal that p53 activation plays a crucial role in chemotherapy-induced apoptosis and reducing cell viability. Moreover, decreasing m⁶A methyltransferase (e.g., methyltransferase-like 3, METTL3) expression through chemotherapeutic drug combinations reduced p53 mRNA m⁶A modification. p53 mRNA m⁶A modification blockage induced by S-adenosyl homocysteine or siRNA-mediated METTL3 inhibition enhanced HCC sensitivity to chemotherapy. Importantly, we observed that downregulation of METTL3 and upregulation of p53 expression by oral administration of chemotherapy drugs triggered apoptosis and xenograft tumor growth inhibition in nude mice. Based on these findings, we hypothesize that a METTL3–m⁶A–p53 axis could be a potential target in HCC therapy.

     

m^6 A RNA甲基化修饰异常在肿瘤中的作用

N6-甲基腺嘌呤(N6-methyladenosine,m6A)是真核生物信使RNA(messenger RNA,mRNA)含量最多的化学修饰之一。m6A修饰主要由m6A甲基转移酶(methyltransferase)催化,m6A去甲基酶(demethylase)去除,并由m6A结合蛋白(binding protein)识别。它广泛参与调控mRNA剪接、加工、翻译和降解等生命周期的各个阶段,且与肥胖和肿瘤等多种疾病及异常的生理功能相关。近年的研究发现,肿瘤中m6A相关蛋白质(METTL3/14、WTAP、FTO、ALKBH5、YTHDFs)的异常表达,引发m6A甲基化的失调,调控致癌基因和抑癌基因的表达参与肿瘤的发生与发展,并与患者预后不良密切相关。随着RNA免疫沉淀测序技术与高通量测序技术和液相色谱等检测技术的快速发展,有关m6A在肿瘤发生发展中的作用机制研究的进展迅猛,靶向m6A也成为肿瘤临床治疗的新方向。本文重点对m6A RNA甲基化相关因子在癌症发生发展中的作用及机制进行综述,总结m6A RNA甲基化检测技术的最新进展,梳理现有文献报道的脱甲基酶抑制剂大黄酸、甲氯芬那酸2(meclofenamic acid2,MA2)和右旋羟戊二酸(R-2-hydroxyglutarate,R-2HG)等在肿瘤靶向治疗中的运用,为以m6A RNA甲基化为切入点的肿瘤防治研究提供思路与理论参考。     

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m¹A), N6-methyladenosine (m⁶A), pseudouridine, and 5-methylcytidine (m⁵C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m⁵C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m¹A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m⁶A antibody confirmed the demethylation of m⁶A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m⁶A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.     

The role of RNA adenosine demethylases in the control of gene expression

RNA modifications are being recognized as an essential factor in gene expression regulation. They play essential roles in germ line development, differentiation and disease. In eukaryotic mRNAs, N⁶-adenosine methylation (m⁶A) is the most prevalent internal chemical modification identified to date. The m⁶A pathway involves factors called writers, readers and erasers. m⁶A thus offers an interesting concept of dynamic reversible modification with implications in fine-tuning the cellular metabolism. In mammals, FTO and ALKBH5 have been initially identified as m⁶A erasers. Recently, FTO m⁶A specificity has been debated as new reports identify FTO targeting N⁶,2′-O-dimethyladenosine (m⁶A_m). The two adenosine demethylases have diverse roles in the metabolism of mRNAs and their activity is involved in key processes, such as embryogenesis, disease or infection. In this article, we review the current knowledge of their function and mechanisms and discuss the existing contradictions in the field. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.