杨莹^1,2,3, 陈宇晟^1,3, 孙宝发^1,2,3, 杨运桂^,1,2,3 1. 中国科学院北京基因组研究所,中国科学院精准基因组医学重点实验室,北京 100101
2. 中国科学院干细胞创新研究院,北京 100101
3. 中国科学院大学,北京 100049

RNA methylation: regulations and mechanisms

Ying Yang^1,2,3, Yusheng Chen^1,3, Baofa Sun^1,2,3, Yungui Yang^,1,2,3 1. CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China
2. Institute of Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China
3. University of Chinese Academy of Sciences, Beijing 100049, China

通讯作者: 杨运桂,博士,研究员,研究方向：RNA表观转录组学。E-mail: ygyang@big.ac.cn

编委: 宋旭
收稿日期:2018-06-29修回日期:2018-10-8网络出版日期:2018-11-20

基金资助:

国家重点研发计划项目(973计划).2016YFC0900300 国家自然科学基金项目.31500659 国家自然科学基金项目.31770872 中国科学院青年创新促进会项目.CAS 2018133

Received:2018-06-29Revised:2018-10-8Online:2018-11-20

Fund supported:

Supported by the Ministry of Science and Technology of China.2016YFC0900300 the National Natural Science Foundation of China .31500659 the National Natural Science Foundation of China .31770872 the Youth Innovation Promotion Association.CAS 2018133

作者简介 About authors
杨莹,博士,副研究员,研究方向：RNA表观转录组学E-mail:yingyang@big.ac.cn。






摘要
表观遗传学修饰包括DNA、RNA和蛋白质的化学修饰,基于非序列改变所致基因表达和功能水平变化。近年来,在DNA和蛋白质修饰基础上,可逆RNA甲基化修饰研究引领了第3次表观遗传学修饰研究的浪潮。RNA存在100余种化学修饰,甲基化是最主要的修饰形式。鉴定RNA甲基化修饰酶及研发其转录组水平高通量检测技术,是揭示RNA化学修饰调控基因表达和功能规律的基础。本文主要总结了近年来本课题组与合作团队及国内外同行在RNA甲基化表观转录组学研究中取得的主要前沿进展,包括发现了RNA去甲基酶、甲基转移酶和结合蛋白,揭示RNA甲基化修饰调控RNA加工代谢,及其调控正常生理和异常病理等重要生命进程。这些系列研究成果证明RNA甲基化修饰类似于DNA甲基化,具有可逆性,拓展了RNA甲基化表观转录组学研究新领域,完善了中心法则表观遗传学规律。
关键词： 表观转录组学;RNA甲基化;RNA加工;6-甲基腺嘌呤;5-甲基胞嘧啶

Abstract
Epigenetic modifications include the chemical modifications on DNA, RNA and proteins characterized by altered gene expression and function without any changes in the gene sequence. In addition to well-established DNA and protein epigenetic modifications, the reversible RNA methylation has led the third wave of studies in the epigenetic field. RNA has more than 100 chemical modifications, among which methylation is the major type. The identification of catalyzing enzymes for RNA methylation and the development of high-throughput detection technologies for RNA modification at the transcriptomic level are the prerequisites for revealing the regulatory role of RNA methylation in gene expression and biological functions. In this review, we summarize the recent frontier in RNA methylation-mediated epitranscriptomics from our and other laboratories, with emphasis on the discoveries of RNA modification demethylase , methyltransferase and binding protein as well as the illustration of regulatory roles of RNA methylation modification in hematopoietic stem cell differentiation, spermatogenesis, brain development and other pivotal life processes. These findings have shown that RNA methylation is just as reversible as DNA methylation, and opened up a novel field in RNA methylation-mediated epitranscriptomics, which appends a new layer of epigenetic regulation to the central genetic dogma.
Keywords：epitranscriptomics;RNA methylation;RNA processing;N ⁶-methyladenosine (m ⁶A) ;5-methylcytosine (m ⁵C)


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本文引用格式
杨莹, 陈宇晟, 孙宝发, 杨运桂. RNA甲基化修饰调控和规律[J]. 遗传, 2018, 40(11): 964-976 doi:10.16288/j.yczz.18-175
Ying Yang, Yusheng Chen, Baofa Sun, Yungui Yang. RNA methylation: regulations and mechanisms[J]. Hereditas(Beijing), 2018, 40(11): 964-976 doi:10.16288/j.yczz.18-175


RNA修饰是一种转录后水平的调控方式,目前已鉴定到超过150种RNA修饰,它们广泛分布于信使RNA (messenger mRNA, mRNA)、转运RNA (transfer RNA, tRNA)、核糖体RNA (ribosomal RNA, rRNA)、非编码小RNA (small non-coding RNA)和长链非编码RNA (long non-coding RNA, lncRNA)等各类RNA上。

6-甲基腺嘌呤(N⁶-methyladenosine,即m⁶A)是mRNA上最为常见的一类RNA修饰,它广泛存在于酵母、植物、果蝇以及哺乳动物等各类真核生物中。虽然早在19世纪70年代就已鉴定到了RNA上的m⁶A存在,但其具体的分子功能却一直处于未知阶段。2011年以来,随着RNA m⁶A第一个去甲基酶FTO (fat mass and obesity-associated protein)的鉴定及抗体富集和高通量测序技术的发展,转录组水平m⁶A分布图谱的精确绘制得以实现,超过25%的转录本上被鉴定到10 000个以上的m⁶A峰,这些m⁶A具有保守修饰基序RRACH (R表示A或G,H表示A、U或C),并且分布富集于长外显子、终止密码子附近以及3°非翻译区(3°untranslated region, 3°UTR)。转录本m⁶A的修饰水平受甲基转移酶(编码器)、结合蛋白(读码器)和去甲基化酶(消码器)的动态调控。近年来以m⁶A为代表的RNA修饰研究成果,证明了RNA修饰动态可逆性调控RNA代谢和加工过程及干细胞定向分化等重要生物学功能^{[1, 2]}。

5-甲基胞嘧啶(5-methylcytosine, m⁵C)是近年来引起广泛关注的另外一种新的修饰类型。m⁵C最早发现于tRNA和rRNA中,mRNA中的m⁵C修饰也已被发现40余年,但对于mRNA中m⁵C修饰的特征及功能目前并不完全清楚。最初基于重亚硫酸盐处理结合转录组测序的研究发现HeLa细胞中上千个mRNA上的m⁵C修饰位点^[3]。基于m⁵C抗体免疫沉淀结合重亚硫酸盐测序的研究也鉴定到了古生菌mRNA中的m⁵C修饰位点,及其保守序列AU (m⁵C) GANGU^[4]。近期,全转录组水平的m⁵C分布特征及功能研究表明,m⁵C具有物种和组织细胞特异性^[5,6,7]。其中,m⁵C甲基转移酶NSUN2 (NOP2/Sun RNA methyltransferase family member 2)及结合蛋白ALYREF (Aly/REF export factor)的发现,也充分证明了RNA m⁵C修饰具有动态可逆性并且能够调控RNA代谢和加工过程及重要的生物学功能。

1 RNA修饰相关蛋白

1.1 RNA去甲基酶(Demethylase)

1.1.1 m⁶A去甲基化酶FTO

19世纪70年代Desrosiers等^[8]发现mRNA中存在m⁶A修饰,但RNA修饰研究一直滞后于DNA修饰研究,究其原因,RNA修饰研究中悬而未决的核心问题是RNA修饰是否调控基因表达,是否具有动态性和可逆性?通过体外模拟生理环境下的去甲基化反应条件,将肥胖基因FTO的野生型和突变型蛋白分别与甲基化底物孵育,利用质谱和高效液相技术,对多种甲基化形式进行探索,最终发现FTO对单链RNA上的m⁶A具有去甲基化功能;同时,在细胞内经高效液相色谱-串联质谱联用(liquid chromatography-tandem mass chromatography, LC-MS/ MS)实验验证发现,FTO基因敲低细胞中mRNA中的m⁶A水平升高,而FTO过表达细胞中mRNA中的m⁶A水平降低。通过免疫荧光实验证实,FTO在细胞核中呈点状分布。通过与各种细胞核内亚细胞器标志分子共染,发现FTO与核小斑标志分子SC35 (splicing component, 35 KDa)、U4/U6.U5 snRNA相关蛋白SART1 (U4/U6.U5 tri-snRNP-associated protein 1)、转录酶RNA PolⅡ (2位Ser磷酸化)有部分共定位。转录抑制后,类似RNA PolⅡ (2位Ser磷酸化),FTO会聚集到核小斑。这些实验结果证实了FTO催化mRNA上m⁶A修饰的去甲基化^[9]。这一重要发现首次揭示了RNA化学修饰存在可逆性,奠定了RNA甲基化表观转录组学研究新领域的理论基础。

此外,近期研究还发现了FTO的其他底物,包括N⁶,2’-O-二甲基腺嘌呤(N⁶, 2’-O-dimethyladenosine, m⁶A_m )^[10,11,12]和1-甲基腺嘌呤(N¹-methyladenosine, m¹A)^[11]。最新的研究系统阐述了FTO介导的RNA去甲基化,其中,细胞核的FTO介导m⁶A的去甲基化,细胞质中的FTO介导m⁶A_m和m⁶A的去甲基化;此外,FTO还可以结合tRNA,介导tRNA的m¹A的去甲基化并进一步影响蛋白的翻译速率^[11]。

1.1.2 m⁶A去甲基化酶ALKBH5

FTO是二价铁/α-酮戊二酸盐依赖型双加氧酶AlkB家族成员之一,该家族其他成员包括ALKBH1- 8。这些ALKBH成员是否也具有m⁶A去甲基酶活性?整合质谱学、细胞生物学、基因组学、生物信息学和模式生物学等,鉴定到ALKBH5 (alkB homolog 5, RNA demethylase)具有催化m⁶A去甲基化活性。在细胞系中敲低ALKBH5,mRNA的m⁶A水平显著升高;与野生型小鼠睾丸相比,Alkbh5敲除小鼠的睾丸中m⁶A水平升高;这些结果证明ALKBH5类似于FTO,具有催化m⁶A去甲基化活性。通过对雄性Alkbh5敲除小鼠的表型分析,发现雄性小鼠睾丸变小、生精异常、精子活力受损。利用转录组测序(RNA sequencing, RNA-seq)和基因功能聚类分析筛查Alkbh5敲除小鼠的睾丸组织差异表达基因,发现受累基因功能通路涉及精子发生,提示m⁶A去甲基化参与调控小鼠精子发育过程^[13]。

1.2 RNA甲基转移酶(Methyltransferase)

1.2.1 m⁶A甲基转移酶复合物核心组分METTL3、METTL14和WTAP

m⁶A去甲基酶FTO和ALKBH5的发现,为鉴定m⁶A甲基转移酶提供了重要线索。早期研究发现RNA m⁶A甲基转移酶全酶复合物由至少两个亚复合物MT-A (~200 kDa)和MT-B (~800 kDa)组成,但基于鉴定技术和酶学方法的缺乏,只有METTL3 (methyltransferase like 3, ~70 kDa)蛋白被鉴定,而该亚基单独是没有酶活性的^[14,15,16]。因此,具有酶活性的m⁶A甲基转移酶核心组分的鉴定,是研究m⁶A调控功能和作用规律的关键。利用串联亲和沉淀结合质谱分析,发现甲基转移酶复合物中的另外两个组分—WTAP (Wilms’ tumor 1-associating protein)和METTL14 (methyltransferase like 14);结合免疫荧光共聚焦显微镜技术和基于光交联免疫共沉淀技术(photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation, PAR-CLIP)的测序技术,鉴定了WTAP和METTL3在细胞内主要作用底物mRNA具有m⁶A保守基序特征RRACH,发现在WTAP基因敲低的情况下METTL3和METTL4在负责mRNA加工的亚细胞器—核小斑上的定位信号减少及METTL3结合RNA亲和力降低,提示WTAP作为调节亚基,调控m⁶A甲基转移酶催化亚基METTL3/ METTL14复合物定位和结合mRNA;WTAP和METTL3基因敲低导致斑马鱼胚胎组织分化发育异常以及细胞凋亡的增多^[17]。

1.2.2 其他组分

m⁶A甲基转移酶属于多因子功能复合物,除了催化亚基METTL3,国内外多个实验室鉴定到的多个新组分包括METTL14^[17,18,19]、WTAP^{[17, 19~21]}、VIRMA (vir like m⁶A methyltransferase associated,也称KIAA1429)^{[22, 23]}、RBM15 (RNA binding motif protein 15)^[24]和ZC3H13 (zinc finger CCCH-type containing 13)^[25,26,27],这些组分的功能很大程度上是作为调节亚基,调控METTL3在细胞内的活性。METTL3的同源蛋白METTL16 (methyltransferase like 16)通过动态调控U6 snRNA和靶mRNA的m⁶A修饰,从而调节细胞内S-腺苷基甲硫氨酸(S-adenosyl methionine,SAM)水平。METTL16的活性需要UACAGAGAA九聚体和一类特殊的RNA结构^[28]。

1.2.3 miRNA调控m⁶A甲基转移酶活性

m⁶A修饰在mRNA保守序列RRACH中碱基 腺嘌呤(A)位点选择性形成。应用m⁶A抗体免疫共沉淀-高通量测序技术(m⁶A-specific methylated RNA immunoprecipitation with next generation sequencing, MeRIP-seq),可以检测到成熟mRNA中1%~5%的RRACH基序被甲基化,因此这些被甲基化的A位点可能存在选择性机制。利用m⁶A甲基化测序技术分析了4种不同多能性程度的细胞(胚胎干细胞、诱导性多能干细胞、神经干细胞、睾丸支持细胞)中mRNA上的m⁶A修饰图谱,发现m⁶A修饰具有基因和细胞类型特异性并且m⁶A修饰在microRNA (miRNA)靶位点富集。在小鼠神经干细胞(neural stem cell, NSC)和人HeLa细胞中,敲低或过表达负责miRNA生成的核酸内切酶Dicer显著影响了m⁶A水平。在小鼠NSC中过表达或抑制与m⁶A修饰区域互补配对的miRNA,发现miRNA相应靶标位点的m⁶A水平显著升高或降低。利用免疫荧光技术发现Dicer能调控METTL3的核小斑定位;利用PAR-CLIP技术发现Dicer敲低后,METTL3结合的RNA水平急剧下降;利用RNA免疫共沉淀技术(RNA immunoprecipitation, RIP)发现miRNA过表达或敲低显著增加或降低了METTL3结合的miRNA靶标mRNA的水平,说明miRNA通过调控METTL3结合mRNA的能力来调控其m⁶A甲基转移酶活性。在表达4个Yamanaka因子(Oct4、Sox2、Klf4、c-Myc)的小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEF)中过表达或敲低Mettl3显著提高或降低了m⁶A的水平和重编程效率,以及多能性因子Oct4、Sox2、Nanog的表达,说明m⁶A修饰促进体细胞重编程为多能性细胞^[29]。这些研究结果揭示了miRNA通过序列互补调控mRNA甲基化修饰形成这一全新的作用机制及m⁶A修饰调控细胞重编程命运重要功能。

1.2.4 m⁵C甲基转移酶NSUN2

除了m⁶A修饰,RNA还存在m⁵C修饰,其在tRNA、rRNA和mRNA等各类RNA中均有分布。尽管在19世纪70年代就已在mRNA上鉴定到m⁵C的存在^{[8, 30]},然而受限于检测技术,直到近年来随着高通量测序技术的发展,m⁵C研究才取得了进展。为揭示mRNA上m⁵C修饰的分布规律和生物学功能,通过优化RNA m⁵C单碱基测序及分析方法,绘制了人类HeLa细胞系及小鼠6个组织的m⁵C转录组分布图谱,发现m⁵C在mRNA具有物种保守性,显著富集于翻译起始密码子下游以及CG富集区域;而在小鼠不同组织中,m⁵C修饰具有组织特异性和发育动态性,提示m⁵C在发育和分化过程中扮演重要调控角色。为鉴定mRNA的m⁵C甲基转移酶,运用超高效液相色谱-三重四级杆-多重反应检测质谱(ultra-high performance liquid chromatography-triple quadrupole mass spectrometry with multiple-reaction monitoring, UHPLC-QQQ-MRM-MS/MS)技术鉴定并发现了NSUN2是mRNA特异的m⁵C甲基转移酶,其催化活性依赖于C271 (Cysteine 271)和C321 (Cysteine 321)两个半胱氨酸位点;高通量测序分析结果也表明,NSUN2敲低能够显著降低mRNA m⁵C的修饰水平^[5]。

1.3 RNA甲基化修饰结合蛋白

1.3.1 m⁶A结合蛋白YTHDC1

m⁶A调控mRNA加工和代谢功能很大程度上依赖于其结合蛋白的有效识别。转录组结合MeRIP-seq分析表明,FTO表达降低可导致m⁶A的水平升高,分化各时期的m⁶A修饰以及FTO介导的m⁶A修饰在5′和3′剪接位点相邻的外显子区域显著富集,调节脂肪前体细胞分化,提示m⁶A修饰可能作为一种新顺式元件调控mRNA的剪接^[31]。那么细胞核内m⁶A修饰如何调控mRNA选择性剪接?通过免疫共沉淀结合质谱分析技术,发现定位于细胞核内的m⁶A结合蛋白YTHDC1 (YTH domain containing 1)与剪接因子SRSF3 (serine and arginine rich splicing factor 3)和SRSF10 (serine and arginine rich splicing factor 10)发生相互作用,提示m⁶A通过YTHDC1调控mRNA选择性剪接。运用RNA-seq分析,发现YTHDC1、METTL3和SRSF3敲低后,转录本外显子的保留水平在总体上是降低的,而SRSF10敲低后则相反。结合光交联增强型免疫共沉淀及测序技术(PAR-CLIP-seq)进一步分析,发现有m⁶A修饰的外显子更倾向于被保留。剪接因子SRSF3和SRSF10能够与YTHDC1直接相互作用,并且竞争性的结合YTHDC1。YTHDC1或METTL3敲低后,SRSF3的RNA结合能力以及蛋白的核内定位信号明显减弱,而SRSF10则呈现相反趋势,说明YTHDC1招募SRSF3到RNA的结合位点,同时抑制SRSF10到RNA结合位点,揭示了m⁶A在细胞核内通过调控选择性剪接的方式影响转录本^[32]。

此外,近期一项研究工作表明,YTHDC1可与SRSF3及RNA出核因子NXF1 (nuclear RNA export factor 1)相互作用,促进m⁶A修饰的mRNA的出核^[33]。该研究进一步拓展了YTHDC1介导的m⁶A对mRNA代谢的调控作用。

近期研究对于HNRNP家族成员HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2/B1)是否是m⁶A结合蛋白还存在争议。Alarcón等^[34]发现HNRNPA2B1可直接结合m⁶A并且与METTL3协同调控可变剪接事件以及miRNA前体的加工。而Wu等^[35]基于蛋白结构的分析发现了一种由HNRNPA2B1介导的“m⁶A开关”而非直接结合的机制。此外,其他两种HNRNP蛋白—HNRNPC (heterogeneous nuclear ribonucleoprotein C)^[36]和HNRNPG (heterogeneous nuclear ribonucleoprotein G)^[37],也可调控m⁶A修饰的RNA转录本的加工。其中,m⁶A作为一种结构开关改变RNA的结构,从而调控HNRNPC和HNRNPG对转录本的结合。

1.3.2 m⁶A结合蛋白YTHDF2

2014年,研究人员首次报道了m⁶A的结合蛋白YTHDF2 (YTH domain family, member 2)可介导m⁶A修饰的mRNA的降解。正常条件下,YTHDF2可与脱腺苷酸酶复合物及脱帽复合物蛋白共定位,并且将其靶基因的转录本带入降解小体^[38]。随后的研究进一步表明,YTHDF2通过招募CCR4-NOT脱腺苷酸复合物从而加速m⁶A修饰的转录本的降解^[39]。

1.3.3 m⁶A结合蛋白YTHDF1

YTHDF1 (YTH domain family, member 1)最初被发现可结合到m⁶A修饰的转录本的终止密码子附近,其整体分布与m⁶A修饰非常相似。此外,YTHDF1可与翻译起始复合物直接作用,从而促进m⁶A修饰的RNA底物的翻译效率^[40]。

1.3.4 m⁶A结合蛋白YTHDF3

定位在细胞质的m⁶A结合蛋白包括YTHDF1、YTHDF2、YTHDF3 (YTH domain family, member 3)和YTHDC2 (YTH domain containing 2)。通过串联亲和沉淀结合质谱技术以及GST-Pull down实验,鉴定出YTHDF3和YTHDF1与核糖体40S小亚基和60S大亚基蛋白相互作用。利用新蛋白合成实验,发现YTHDF3可以促进翻译效率,回补实验证明这种现象只能被野生型的YTHDF3蛋白回补,而不能被m⁶A结合关键位点突变体(Tryptophan 438, W438; Tryptophan 492, W492) YTHDF3蛋白回补。PAR- CLIP-seq结合生物信息学分析发现,YTHDF1和YTHDF3结合基序相似,结合位点都主要位于3′UTR。YTHDF3可以促进YTHDF1和YTHDF3共有靶基因的翻译效率,提示YTHDF3和YTHDF1协同调控mRNA翻译效率,揭示了m⁶A在细胞质内读码器YTHDF3调控mRNA的翻译效率的新机制^[41]。

此外,通过与YTHDF2直接作用,YTHDF3还可以介导mRNA降解^[42]。

1.3.5 m⁶A结合蛋白YTHDC2

YTHDC2是YTH家族中分子量最大的一个成员,也倾向于结合m⁶A修饰的保守基序,并增强其底物的翻译效率或降低底物的丰度^{[43,44,45,46]}。此外,YTHDC2还与小鼠精子发生密切相关^[43]。

1.3.6 其他m⁶A结合蛋白

在酵母中,YTH家族的另一成员Mrb1 (methylated RNA binding protein 1)也被报道可结合m⁶A修饰的RNA^[47]。此外,一些基于RNA pull-down的研究检测到了其他的m⁶A结合蛋白,包括ELAVL1 (ELAV-like protein 1)^{[18, 48, 49]}、FMR1 (fragile X mental retardation 1)^{[50, 51]}、LRPPRC (leucine rich pentatricopeptide repeat containing)^[51]以及IGF2BP家族蛋白(insulin like growth factor 2 mRNA binding proteins)^[52]。然而,这些蛋白是否直接结合m⁶A,以及他们是否是m⁶A结合复合物的一部分,还有待深入研究。

1.3.7 m⁵C结合蛋白ALYREF

为了鉴定m⁵C的结合蛋白,设计了包含和不包含m⁵C两种RNA寡聚核苷酸底物,通过寡聚核苷酸富集联合蛋白质谱技术,鉴定到mRNA出核功能复合物组分ALYREF蛋白能够结合m⁵C修饰的RNA寡聚核苷酸。结合凝胶迁移实验(electrophoretic mobility shift assay,EMSA)及RIP结合HPLC和测序技术,发现ALYREF的K171 (Lysine 171)突变能够显著降低其结合m⁵C的能力,进而降低其与RNA的结合能力。利用荧光原位杂交(fluorescence in situ hybridization,FISH)技术,发现mRNA出核效率随着NSUN2和ALYREF的敲低而降低。通过回补实验,发现只有NSUN2和ALYREF的野生型蛋白能够分别回补NSUN2和ALYREF敲低而导致的mRNA出核降低,提示m⁵C在mRNA出核过程中具有重要的调控作用^[5]。

2 RNA甲基化修饰的生物学功能

2.1 RNA甲基化修饰m⁶A调控造血干细胞定向分化

METTL3介导的m⁶A修饰在生物节律、DNA损伤应答、干细胞的自我更新和多能性调控、母源-合子转换、果蝇神经功能调节与性别决定、小鼠早期胚胎发育等真核生物的各种生物学过程和个体发育中发挥着非常重要的作用^{[29, 31, 53~60]}。RNA甲基修饰酶和结合蛋白及其参与RNA代谢加工的发现,提示RNA甲基化修饰具有重要生物学功能。脊椎动物中,造血干细胞最初由特化的生血内皮通过内皮-造血转化过程(endothelial-to-haematopoietic transition, EHT)产生于胚胎期主动脉-性腺-中肾区,随后向血组织迁移并进行扩增,向胸腺迁移发育为淋系细胞,最后向骨髓(小鼠和人)或肾髓(斑马鱼)迁移以维持终生造血。通过m⁶A抗体进行MeRIP-seq和m⁶A单碱基分辨率的miCLIP-seq测序技术,绘制了斑马鱼胚胎发育的m⁶A修饰精细图谱,发现m⁶A通过特异性修饰内皮-造血转化过程中的关键基因notch1a,招募结合蛋白YTHDF2降解mRNA,抑制了Notch信号通路,使得内皮-造血转化过程有序发生,促进内皮细胞转化为造血干/祖细胞(haematopoietic stem/ progenitor cells, HSPCs)。在mettl3缺陷的斑马鱼胚胎中,m⁶A修饰水平显著降低,notch1a无法正常降解使得Notch信号通路持续激活,最终导致内皮细胞无法正常转化为造血干/组细胞^[61]。此外,Mettl3敲除小鼠胚胎中也表现出相似的表型^[62]。这些结果揭示mRNA m⁶A甲基化修饰在脊椎动物造血干细胞命运决定中的调控机制,阐释RNA的表观修饰在血液发育中的关键作用。

2.2 RNA甲基化修饰m⁶A调控精子发生

由于Mettl3敲除会导致小鼠早期胚胎致死^[56],Mettl3条件敲除介导的m⁶A修饰在哺乳动物体内组织发育中的生物学功能还不清楚。利用CRISPR/ Cas9系统介导的同源重组技术和Cre-loxP系统成功地建立了生殖细胞(Vasa-Cre)中特异性敲除Mettl3的小鼠(Mettl3^CKO)模型,并利用该模型系统地研究了METTL3介导的m⁶A修饰在小鼠精子发生过程中的重要作用和调控机制。通过H&E(苏木素-伊红)染色、免疫荧光染色、核染色体铺片等研究表明,Mettl3敲除会抑制小鼠精原干细胞分化和减数分裂起始过程,最终导致雄性小鼠不育。通过RNA-seq、m⁶A- miCLIP-seq和生物信息学分析发现,Mettl3缺失会导致m⁶A水平下降,引起与精原干细胞维持和分化、细胞周期和减数分裂相关基因的RNA可变剪接异常和生殖细胞整体基因表达的紊乱,最终导致精子发生过程受阻,说明RNA甲基转移酶METTL3介导的m⁶A修饰在哺乳动物小鼠精子发生过程中的重要生物学功能^[63]。

在小鼠生殖细胞中用Vasa-Cre特异性敲除Mettl3或者Mettl14会导致m⁶A水平下降,引起精原干细胞增殖和分化相关基因转录物的翻译功能失调,精原干细胞逐渐耗尽;利用Stra8-GFPCre同时敲除Mettl3和Mettl14,会破坏精子发生过程中单倍体特异性基因的翻译,最终导致精子形成受阻^[64]。而雄性m⁶A去甲基酶Alkbh5^-/-小鼠也出现睾丸变小、生精异常、精子活力异常等表型;同时在发育至Ⅶ期的生精小管中检测到初级与次级精母细胞数量显著增加,粗线精母细胞与成熟精子的数量明显减少,同时伴有细胞凋亡^[13]。Ythdc2敲除小鼠也发生在精细胞减数分裂前期出现过度的细胞凋亡,从而导致睾丸变小及精子发育异常^[43]。YTHDC2的m⁶A结合能力及其3′→5′解旋酶活性对于维持精子发育过程中减数分裂相关基因的正确表达模式具有重要调控功能^[45]。上述研究提示RNA m⁶A甲基化修饰的动态平衡是配子(精子)发育过程中重要的调控因素。

2.3 RNA甲基化修饰m⁶A调控脑发育

m⁶A修饰在神经系统的发育以及功能的行使中也发挥不可替代的调控作用^{[59, 65, 66]}。m⁶A RNA甲基化修饰在中枢神经系统的发育以及功能行使中发挥重要的调控作用。敲除Ime4基因的果蝇可以出生并存活,但果蝇寿命变短,并表现出明显的行为异常,提示m⁶A修饰的缺失影响果蝇神经系统功能^[59]。在小鼠的中枢神经系统中特异地敲除Mettl14基因会严重影响小鼠大脑皮质的发育^[67]。小鼠中Ythdf2基因缺失导致m⁶A整体水平升高,使得参与神经干细胞分化和神经元轴树突形成的重要因子无法正常进入RNA降解途径,从而导致大脑皮层神经干细胞不能正常地进行不对称分裂,造成神经前体细胞的大量缺失、严重影响神经元的分化,致使小鼠的前脑大脑皮层发育缓慢^[68]。除大脑皮层外,小脑的RNA m⁶A甲基化模式和水平尤为突出。m⁶A甲基化与去甲基化的动态过程贯穿于小脑出生后发育的整个过程,且在低压低氧环境下Alkbh5基因缺失造成参与小脑发育调控进程基因的m⁶A水平紊乱,加快了RNA出核过程,从而导致小脑发育明显滞后^[69]。

通过Cre-loxP系统在小鼠的中枢神经系统中特异性地敲除Mettl3基因,以探究m⁶A甲基化修饰对中枢神经系统发育的影响。在中枢神经系统特异地敲除Mettl3导致小鼠在哺乳期表现出严重的运动功能障碍,并导致死亡。解剖和病理切片检测发现由Mettl3敲除引起的m⁶A甲基化修饰的缺失严重影响大脑皮层和小脑的发育,导致大脑皮层偏薄和小脑发育不良。m⁶A甲基化修饰的缺失引起小脑内颗粒神经元分化和成熟过程中基因表达调控紊乱,导致新生的颗粒神经元大量凋亡^[70],揭示了METTL3介导的m⁶A修饰在哺乳动物中枢神经系统发育中的重要作用。

除了对中枢神经系统发育的影响,m⁶A修饰也参与成体的神经系统调控,神经轴突损伤会促使神经细胞内m⁶A修饰水平提高,进而提高包括轴突再生相关基因在内的一系列基因的蛋白质翻译效率^[70]。

2.4 RNA甲基化修饰m⁶A与RNA代谢

目前越来越多的研究表明,从细胞核内的前体mRNA加工、mRNA表达到细胞质的mRNA翻译和降解,m⁶A几乎参与调控了RNA加工代谢的各个过程。在细胞核内,m⁶A通过结合蛋白YTHDC1招募SRSF3,参与调控前体mRNA的选择性剪接过程^[32];通过调控最后一个外显子的选择性剪接,参与调控腺苷多聚体的多样性^{[71, 72]};而m⁶A修饰酶体系METTL3、ALKBH5和YTHDC1还被发现参与调控mRNA的出核过程^{[13, 33]}。在细胞质中,m⁶A调控翻译的机制十分多样,既有通过YTHDF1-eIF3 (eukaryotic translation initiation factor 3)通路调控mRNA翻译效率的方式^[40],也存在IGF2BP家族蛋白介导的调控过程^[52],并且在应激状态下,m⁶A参与一类不依赖于5′帽子的翻译调控通路^[73];并且其对mRNA稳定性的调控也存在不同的通路,既包括通过YTHDF2将mRNA招募至P小体降解mRNA的调控方式^[38],近期有报道指出IGF2BP (insulin-like growth factor 2 mRNA-binding proteins)家族蛋白通过特异性结合m⁶A修饰mRNA维持其稳定性^[52]。此外,m⁶A还可通过调控RNA的二级结构调控其代谢过程^{[36, 37]}。

2.5 RNA甲基化修饰m⁵C的生物学功能

近年来越来越多的研究表明,mRNA上的m⁵C修饰在RNA加工代谢及正常生理过程中发挥重要的调控作用,包括RNA代谢、干细胞分化及应激反应等过程。对小鼠胚胎干细胞和大脑的研究表明,在干细胞分化不同时期,m⁵C修饰水平和分布明显不同^[7]。在拟南芥中,mRNA上的m⁵C修饰呈现组织特异性,且对植物发育非常重要^{[6, 74]}。拟南芥中的m⁵C甲基转移酶TRM4B突变体植株的根顶端分生组织细胞分裂受阻,从而导致除根较短,并且对氧化应激非常敏感^[6]。

3 结语与展望

在读码器和消码器的共同作用下,RNA m⁶A修饰水平得以实现动态调控,并通过招募不同的结合蛋白实现对RNA加工过程的精细调控。虽然该研究领域还有部分观点存在不确定性,需要在将来投入更多的研究工作来证明。目前通过改造DNA聚合酶实现m⁶A修饰位点错配以检测单碱基精度的m⁶A转录组修饰水平^[75],以及借助纳米孔测序直接检测转录本上鉴定RNA修饰的技术^[76],已成为领域内最热门的研究热点。该技术的实现,将成为阐明m⁶A修饰动态调控机制及其分子功能的核心关键。

对于m⁵C修饰,更为精确的m⁵C检测方法的开发对于高可信度的m⁵C修饰谱的建立至关重要。同样,纳米孔测序对于m⁵C的研究也非常重要。其次,m⁵C的书写器、阅读器和擦除器的研究才刚刚开始,这些方向的发展将为揭示RNA m⁵C修饰的生物学功能提供线索。此外,由m⁵C氧化产生的5-羟甲基- 胞嘧啶(5-hydroxymethylcytosine, hm⁵C)等新型RNA修饰类型的特征及功能也有待未来进行深入研究。

除了m⁶A与m⁵C之外,RNA新型化学修饰也是近期研究的热点。最近研究人员发现mRNA上还存在另外一种新型甲基化修饰——m¹A,研究人员利用特异识别m¹A的抗体来富集含有m¹A的RNA片段并结合高通量测序,获得了全转录组水平的m¹A甲基化谱图,发现m¹A特异富集在5′非翻译区(5′UTR)区域^{[77, 78]};并且,还发展出单碱基分辨率的m¹A检测方法,发现核编码和线粒体编码转录本上存在不同类型的m¹A甲基化组^[79]。目前对于mRNA上m¹A修饰的研究仍然处于起步阶段,例如5′UTR的m¹A甲基转移酶,m¹A修饰参与RNA代谢调控的具体分子机制,m¹A修饰是否像m⁶A修饰一样在多个生物学过程中发挥调控作用等都有待未来进行深入的研究。

综上所述,以m⁶A为代表的RNA修饰在RNA加工及生命过程中扮演着重要的调控作用(表1)。随着研究的深入及先进技术的开发,RNA修饰调控基因表达的生物学过程与机制一定会更加清楚。

Table 1
表1
表1 RNA修饰的主要功能及调控蛋白
Table 1 The functions and regulatory machinery of RNA modifications

修饰类型	主要功能	调节蛋白	参考文献
m6A	鉴定了第一个m6A去甲基化酶FTO	FTO	[9]
	FTO还可以催化m6Am和m1A的去甲基化	FTO	[10~12]
	去甲基化酶FTO介导的m6A修饰可以作为新型顺式元件调控mRNA剪接,及脂肪前体细胞分化	FTO	[31]
	鉴定了第二个m6A去甲基化酶ALKBH5,发现m6A去甲基化参与mRNA出核及小鼠精子发育	ALKBH5	[13]
	鉴定了m6A甲基转移酶复合物的新组分WTAP和METTL14,WTAP作为调节亚基调控催化亚基METTL3/METTL14复合物的定位及底物结合能力	WTAP/METTL3/METTL14	[17]
	发现miRNA通过序列互补调控mRNA甲基化修饰形成机制及m6A调控细胞重编程的重要功能	METTL3	[29]
	Mettl3介导的m6A调控小鼠精子发生过程	METTL3	[63]
	Mettl3介导的m6A调控小鼠小脑发育	METTL3	[70]
	m6A甲基转移酶复合物的组分鉴定,包括METTL14,WTAP,VIRMA,RBM15,ZC3H13,以及METTL16等	METTL14、WTAP、VIRMA、 RBM15、ZC3H13、METTL16	[17~28]
	m6A结合蛋白YTHDC1可与SRSF3及SRSF10直接相互作用,调控mRNA选择性剪接	YTHDC1	[32]
	m6A结合蛋白YTHDC1可与SRSF3及RNA出核因子NXF1相互作用,调控mRNA出核	YTHDC1	[33]
修饰类型	主要功能	调节蛋白	参考文献
m6A	m6A结合蛋白YTHDF1可与翻译起始复合物直接作用,促进m6A修饰的RNA底物的翻译效率	YTHDF1	[40]
	m6A结合蛋白YTHDF2介导m6A修饰的mRNA的降解	YTHDF2	[38, 39]
	m6A调控造血干细胞定向分化	YTHDF2、METTL3	[61, 62]
	m6A结合蛋白YTHDF3可与YTHDF1协同作用调控mRNA翻译	YTHDF3/YTHDF1	[41]
	m6A结合蛋白YTHDF3可与YTHDF2协同作用介导mRNA降解	YTHDF3/YTHDF2	[42]
	m6A结合蛋白YTHDC2调控mRNA翻译或降解,以及小鼠精子发生过程	YTHDC2	[43~46]
	m6A结合蛋白IGF2BP1/2/3介导mRNA稳定性及翻译	IGF2BP1/2/3	[52]
	其他可能的m6A结合蛋白	Mrb1, ELAVL1、FMR1、LRPPRC	[18, 47~51]
m5C	发现了mRNA m5C的分布规律,鉴定了mRNA m5C的主要甲基转移酶NSUN2和第一个结合蛋白ALYREF,及其调控mRNA出核的分子机制	NSUN2、ALYREF	[5]
	拟南芥mRNA m5C修饰调控组织发育,甲基转移酶为TRM4B	TRM4B	[6]
m1A	揭示了全转录组水平的m1A甲基化图谱		[77, 78]
	发展了单碱基分辨率的m1A测序方法,发现核编码及线粒体编码转录本上不同类型的m1A甲基化组	ALKBH3	[79]

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The authors have declared that no competing interests exist.

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[1] Yang Y, Hsu PJ, Chen YS, Yang YG . Dynamic transcriptomic m ⁶A decoration: writers, erasers, readers and functions in RNA metabolism
Cell Res, 2018,28(6):616-624.
URLPMID:29789545 [本文引用: 1]
Abstract N 6 -methyladenosine (m 6 A) is a chemical modification present in multiple RNA species, being most abundant in mRNAs. Studies on enzymes or factors that catalyze, recognize, and remove m 6 A have revealed its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. This review describes the current understanding of the m 6 A modification, particularly the functions of its writers, erasers, readers in RNA metabolism, with an emphasis on its role in regulating the isoform dosage of mRNAs.

[2] Frye M, Harada BT, Behm M, He C . RNA modifications modulate gene expression during development
Science, 2018,361(6409):1346-1349.
URL [本文引用: 1]

[3] Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T . Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA
Nucleic Acids Res, 2012,40(11):5023-5033.
URLPMID:22344696 [本文引用: 1]
The modified base 5-methylcytosine (m5C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m5C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m5C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10 275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.

[4] Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R . Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m ⁵C within archaeal mRNAs
PLoS Genet, 2013,9(6):e1003602.
URLPMID:23825970 [本文引用: 1]
Author Summary Ribonucleic acids are universally used to express genetic information in the form of gene transcripts. Although we envision RNA as a mere copy of the DNA four-base code, modification of specific RNA bases can expand the information code. Such modifications are abundant in transfer RNA (tRNA) and ribosomal RNA (rRNA), where they contribute to translation fidelity and ribosome assembly. Recent studies in eukaryotes have shown that mRNA modifications such as RNA-editing (conversion of an adenosine base to inosine), N6-adenine methylation (m6A), and 5-methylcytidine (m5C) can change the coding sequence, alter splicing patterns, or change RNA stability. However, no mRNA modifications in bacteria or archaea have been documented to date. We have used an approach that enables mapping of the m5C modifications across all expressed genes in a given organism. Applying this approach on model bacterial, archaeal, and fungal microorganisms enabled us to reveal the modified RNA bases in these organisms, and to provide an accurate and sensitive map of these modifications. In archaea, we documented multiple genes whose mRNAs are subject to RNA modification, suggesting that similar to eukaryotes, these organisms may utilize mRNA modifications as a mechanism for gene regulation.

[5] Yang X, Yang Y, Sun BF, Chen YS, Xu JW, Lai WY, Li A, Wang X, Bhattarai DP, Xiao W, Sun HY, Zhu Q, Ma HL, Adhikari S, Sun M, Hao YJ, Zhang B, Huang CM, Huang N, Jiang GB, Zhao YL, Wang HL, Sun YP, Yang YG . 5-methylcytosine promotes mRNA export-NSUN2 as the methyltransferase and ALYREF as an m ⁵C reader
Cell Res, 2017,27(5):606-625.
URL [本文引用: 3]

[6] David R, Burgess A, Parker B, Li J, Pulsford K, Sibbritt T, Preiss T, Searle IR . Transcriptome-wide mapping of RNA 5-methylcytosine in Arabidopsis mRNAs and noncoding RNAs
Plant Cell, 2017,29(3):445-460.
URLPMID:28062751 [本文引用: 3]
Posttranscriptional methylation of RNA cytosine residues to 5-methylcytosine (m5C) is an important modification with diverse roles, such as regulating stress responses, stem cell proliferation, and RNA metabolism. Here, we used RNA bisulfite sequencing for transcriptome-wide quantitative mapping of m5C in the model plant Arabidopsis thaliana. We discovered more than a thousand m5C sites in Arabidopsis mRNAs, long noncoding RNAs, and other noncoding RNAs across three tissue types (siliques, seedling shoots, and roots) and validated a number of these sites. Quantitative differences in methylated sites between these three tissues suggest tissue-specific regulation of m5C. Perturbing the RNA m5C methyltransferase TRM4B resulted in the loss of m5C sites on mRNAs and noncoding RNAs and reduced the stability of tRNAAsp(GTC). We also demonstrate the importance of m5C in plant development, as trm4b mutants have shorter primary roots than the wild type due to reduced cell division in the root apical meristem. In addition, trm4b mutants show increased sensitivity to oxidative stress. Finally, we provide insights into the targeting mechanism of TRM4B by demonstrating that a 50-nucleotide sequence flanking m5C C3349 in MAIGO5 mRNA is sufficient to confer methylation of a transgene reporter in Nicotiana benthamiana.

[7] Amort T, Rieder D, Wille A, Khokhlova-Cubberley D, Riml C, Trixl L, Jia XY, Micura R, Lusser A . Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain
Genome Biol, 2017,18(1):1.
URLPMID:5225599 [本文引用: 2]
Recent work has identified and mapped a range of posttranscriptional modifications in mRNA, including methylation of the N6 and N1 positions in adenine, pseudouridylation, and methylation of carbon 5 in cytosine (m5C). However, knowledge about the prevalence and transcriptome-wide distribution of m5C is still extremely limited; thus, studies in different cell types, tissues, and organisms are needed to gain insight into possible functions of this modification and implications for other regulatory processes. We have carried out an unbiased global analysis of m5C in total and nuclear poly(A) RNA of mouse embryonic stem cells and murine brain. We show that there are intriguing differences in these samples and cell compartments with respect to the degree of methylation, functional classification of methylated transcripts, and position bias within the transcript. Specifically, we observe a pronounced accumulation of m5C sites in the vicinity of the translational start codon, depletion in coding sequences, and mixed patterns of enrichment in the 3 UTR. Degree and pattern of methylation distinguish transcripts modified in both embryonic stem cells and brain from those methylated in either one of the samples. We also analyze potential correlations between m5C and micro RNA target sites, binding sites of RNA binding proteins, andN6-methyladenosine. Our study presents the first comprehensive picture of cytosine methylation in the epitranscriptome of pluripotent and differentiated stages in the mouse. These data provide an invaluable resource for future studies of function and biological significance of m5C in mRNA in mammals. The online version of this article (doi:10.1186/s13059-016-1139-1) contains supplementary material, which is available to authorized users.

[8] Desrosiers R, Friderici K, Rottman F . Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells
Proc Natl Acad Sci USA, 1974,71(10):3971-3975.
URLPMID:4372599 [本文引用: 2]
The poly(A) tract found in eukaryotic mRNA was used to study methylation in mRNA obtained from Novikoff hepatoma cells. Methyl labeling of RNA was achieved with L-[methyl-3H]methionine under conditions that suppress radioactive incorporation into the purine ring. RNA that contains a poly(A) segment was obtained from polysomal RNA by chromatography on oligo(dT)-cellulose. Sucrose density gradient centrifugation of this RNA revealed a pattern expected for mRNA. The composition of the methyl-labeld nucleosides in the RNA was analyzed after complete enzymatic degradation to nucleosides. By use of DEAE-cellulose (borate) chromatography, which separates 2′-O-methylnucleosides from normal and base-methylated nucleosides, about 50% of the radioactivity was recovered in the 2′-O-methylnucleoside fraction and 50% in the base-methylnucleoside fraction. High-speed liquid chromatography (Aminex A-5) of the 2′-O-methylnucleoside fraction produced four peaks coincident with the four 2′-O-methylnucleoside standards. Analysis of the base-methylnucleoside fraction revealed a unique pattern. While ribosomal RNA and tRNA possessed complex base-methylnucleoside patterns, the distribution in mRNA was quite simple, consisting predominantly of N6-methyladenosine. These results demonstrate a unique distribution of methylated nucleosides in mRNA. By analogy to ribosomal RNA synthesis, the presence of methylnucleosides in mRNA may reflect a cellular mechanism for the selective processing of certain mRNA sequences.

[9] Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG, He C . N ⁶-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO
Nat Chem Biol , 2011,7(12):885-887.
URLPMID:22002720 [本文引用: 1]
Abstract We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO.

[10] Mauer J, Luo X, Blanjoie A, Jiao X, Grozhik AV, Patil DP, Linder B, Pickering BF, Vasseur JJ, Chen Q, Gross SS, Elemento O, Debart F, Kiledjian M, Jaffrey SR . Reversible methylation of m ⁶A_m in the 5' cap controls mRNA stability
Nature, 2017,541(7637):371-375.
URLPMID:28002401 [本文引用: 1]
Abstract Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N 6 ,2'-O-dimethyladenosine (m 6 A m ), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m 6 A m we find that m 6 A m -initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m 6 A m -initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m 6 A m is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m 6 A m rather than N 6 -methyladenosine (m 6 A), and reduces the stability of m 6 A m mRNAs. Together, these findings show that the methylation status of m 6 A m in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

[11] Wei J, Liu F, Lu Z, Fei Q, Ai Y, He PC, Shi H, Cui X, Su R, Klungland A, Jia G, Chen J, He C . Differential m ⁶A, m ⁶A_m, and m ¹A demethylation mediated by FTO in the cell nucleus and cytoplasm
Mol Cell, 2018,71(6):973-985.
URL [本文引用: 3]

[12] Su R, Dong L, Li C, Nachtergaele S, Wunderlich M, Qing Y, Deng X, Wang Y, Weng X, Hu C, Yu M, Skibbe J, Dai Q, Zou D, Wu T, Yu K, Weng H, Huang H, Ferchen K, Qin X, Zhang B, Qi J, Sasaki AT, Plas DR, Bradner JE, Wei M, Marcucci G, Jiang X, Mulloy JC, Jin J, He C , Chen J. R-2HG exhibits anti-tumor activity by targeting FTO/ m ⁶A/MYC/CEBPA signaling
Cell , 2018, 172(1-2):90- 105. e23.
URLPMID:29249359 [本文引用: 1]
Abstract R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N 6 -methyladenosine (m 6 A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m 6 A/MYC/CEBPA signaling.

[13] Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vagbo CB, Shi Y, Wang WL, Song SH, Lu Z, Bosmans RP, Dai Q, Hao YJ, Yang X, Zhao WM, Tong WM, Wang XJ, Bogdan F, Furu K, Fu Y, Jia G, Zhao X, Liu J, Krokan HE, Klungland A, Yang YG, He C . ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility
Mol Cell, 2013,49(1):18-29.
URLPMID:23177736 [本文引用: 3]
Abstract N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells. Copyright 2013 Elsevier Inc. All rights reserved.

[14] Tuck MT . Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues
Biochem J, 1992,288(Pt1):233-240.
URLPMID:1445268 [本文引用: 1]
Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5' cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.

[15] Bokar JA, Rath-Shambaugh ME, Ludwiczak R, Narayan P, Rottman F . Characterization and partial purification of mRNA N ⁶-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex
J Biol Chem , 1994,269(26):17697-17704.
[本文引用: 1]

[16] Bujnicki JM, Feder M, Radlinska M, Blumenthal RM . Structure prediction and phylogenetic analysis of a functionally diverse family of proteins homologous to the MT-A70 subunit of the human mRNA: m ⁶A methylransferase
J Mol Evol, 2002,55(4):431-444.
URLPMID:12355263 [本文引用: 1]
MT-A70 is the S-adenosylmethionine-binding subunit of human mRNA:m(6)A methyltransferase (MTase), an enzyme that sequence-specifically methylates adenines in pre-mRNAs. The physiological importance yet limited understanding of MT-A70 and its apparent lack of similarity to other known RNA MTases combined to make this protein an attractive target for bioinformatic analysis. The sequence of MT-A70 was subjected to extensive in silico analysis to identify orthologous and paralogous polypeptides. This analysis revealed that the MT-A70 family comprises four subfamilies with varying degrees of interrelatedness. One subfamily is a small group of bacterial DNA:m(6)A MTases. The other three subfamilies are paralogous eukaryotic lineages, two of which have not been associated with MTase activity but include proteins having substantial regulatory effects. Multiple sequence alignments and structure prediction for members of all four subfamilies indicated a high probability that a consensus MTase fold domain is present. Significantly, this consensus fold shows the permuted topology characteristic of the beta class of MTases, which to date has only been known to include DNA MTases. [References: 53]

[17] Ping XL, Sun BF, Wang L, Xiao W, Yang X, Wang WJ, Adhikari S, Shi Y, Lv Y, Chen YS, Zhao X, Li A, Yang Y, Dahal U, Lou XM, Liu X, Huang J, Yuan WP, Zhu XF, Cheng T, Zhao YL, Wang X, Rendtlew Danielsen JM, Liu F, Yang YG . Mammalian WTAP is a regulatory subunit of the RNA N ⁶-methyladenosine methyltransferase
Cell Res , 2014,24(2):177-189.
[本文引用: 3]

[18] Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, Zhao JC . N ⁶-methyladenosine modification destabilizes developmental regulators in embryonic stem cells
Nat Cell Biol , 2014,16(2):191-198.
URLPMID:20202062020202020202020 [本文引用: 2]
N(6)-methyladenosine (m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes. m(6)A modification is involved in cell fate determination in yeast and embryo development in plants. Its mammalian function remains unknown but thousands of mammalian mRNAs and long non-coding RNAs (lncRNAs) show m(6)A modification and m(6)A demethylases are required for mammalian energy homeostasis and fertility. We identify two proteins, the putative m(6)A MTase, methyltransferase-like 3 (Mettl3; ref. ), and a related but uncharacterized protein Mettl14, that function synergistically to control m(6)A formation in mammalian cells. Knockdown of Mettl3 and Mettl14 in mouse embryonic stem cells (mESCs) led to similar phenotypes, characterized by lack of m(6)A RNA methylation and lost self-renewal capability. A large number of transcripts, including many encoding developmental regulators, exhibit m(6)A methylation inversely correlated with mRNA stability and gene expression. The human antigen R (HuR) and microRNA pathways were linked to these effects. This gene regulatory mechanism operating in mESCs through m(6)A methylation is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.

[19] Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng X, Dai Q, Chen W, He C . A METTL3- METTL14 complex mediates mammalian nuclear RNA
N ⁶-adenosine methylation. Nat Chem Biol , 2014,10(2):93-95.
[本文引用: 2]

[20] Zhong S, Li H, Bodi Z, Button J, Vespa L, Herzog M, Fray RG . MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor
Plant Cell, 2008,20(5):1278-1288.
URL
N6-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N6-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS9 TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N6-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

[21] Agarwala SD, Blitzblau HG, Hochwagen A, Fink GR . RNA methylation by the MIS complex regulates a cell fate decision in yeast
Plos Genet, 2012,8(2):e1002732.
URLPMID:3369947 [本文引用: 1]
For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions.

[22] Schwartz S, Mumbach MR, Jovanovic M, Wang T, Maciag K, Bushkin GG, Mertins P, Ter-Ovanesyan D, Habib N, Cacchiarelli D, Sanjana NE, Freinkman E, Pacold ME, Satija R, Mikkelsen TS, Hacohen N, Zhang F, Carr SA, Lander ES, Regev A . Perturbation of m ⁶A writers reveals two distinct classes of mRNA methylation at Internal and 5' sites
Cell Rep, 2014,8(1):284-296.
URLPMID:24981863 [本文引用: 1]
N6-methyladenosine (m6A) is a highly abundant modification of mRNA. Schwartz et al. identify and validate a network of proteins required for mRNA methylation in mammalian cells. They define two distinct classes of methylation sites. The majority of sites depend on the identified proteins, are located at internal positions in transcripts, and inversely correlate with mRNA stability. Sites independent of these proteins form at the first transcribed base as part of the cap structure, forming a previously unappreciated layer of transcriptome complexity.

[23] Yue YN, Liu J, Cui XL, Cao J, Luo GZ, Zhang ZH, Cheng T, Gao MS, Shu X, Ma HH, Wang FQ, Wang XX, Shen B, Wang YZ, Feng XH, He C, Liu JZ . VIRMA mediates preferential m ⁶A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation
Cell Discov, 2018,4:10.
URL [本文引用: 1]

[24] Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, Guttman M , Yang YG.. m ⁶A RNA methylation promotes XIST-mediated transcriptional repression
Nature, 2016,537(7620):369-373.
URLPMID:5509218 [本文引用: 1]
The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N-methyladenosine (mA) residues reversible base modification of unknown function in long non-coding RNAs. We show that mA formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the mA-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent mA consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an mA methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of mA-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes mA residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of mA. These data reveal a pathway of mA formation and recognition required for XIST-mediated transcriptional repression.

[25] Guo J, Tang HW, Li J, Perrimon N, Yan D . Xio is a component of the Drosophila sex determination pathway and RNA N ⁶-methyladenosine methyltransferase complex
Proc Natl Acad Sci USA , 2018,115(14):3674-3679.
[本文引用: 1]

[26] Knuckles P, Lence T, Haussmann IU, Jacob D, Kreim N, Carl SH, Masiello I, Hares T, Villaseñor R, Hess D, Andrade-Navarro MA, Biggiogera M, Helm M, Soller M, Bühler M, Roignant JY . Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m ⁶A machinery component Wtap/Fl(2)d
Genes Dev, 2018,32(5-6):415-429.
URL [本文引用: 1]

[27] Wen J, Lv R, Ma H, Shen H, He C, Wang J, Jiao F, Liu H, Yang P, Tan L, Lan F, Shi YG, He C, Shi Y , Diao J. Zc3h13 regulates nuclear RNA m ⁶A methylation and mouse embryonic stem cell self-renewal
Mol Cell, 2018, 69(6): 1028- 1038. e6.
URLPMID:29547716 [本文引用: 1]
N 6 -methyladenosine (m 6 A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export, and translation. However, the precise m 6 A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc-finger protein, plays an important role in modulating RNA m 6 A methylation in the nucleus. We show that knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m 6 A level on mRNA. Upon Zc3h13 knockdown, a great majority of WTAP, Virilizer, and Hakai translocate to the cytoplasm, suggesting thatZc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m 6 A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer, or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer, and Hakai in the nucleus to facilitate m 6 A methylation and to regulate mESC self-renewal.

[28] Pendleton KE, Chen B, Liu K, Hunter OV, Xie Y, Tu BP , Conrad NK. The U6 snRNA m ⁶A methyltransferase METTL16 regulates SAM synthetase intron retention
Cell , 2017, 169(5): 824- 835. e14.
URLPMID:28525753 [本文引用: 1]
Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N 6 -adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3′ UTR. Increasing METTL16 occupancy on the MAT2A 3′ UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

[29] Chen T, Hao YJ, Zhang Y, Li MM, Wang M, Han WF, Wu YS, Lv Y, Hao J, Wang LB, Li A, Yang Y, Jin KX, Zhao X, Li YH, Ping XL, Lai WY, Wu LG, Jiang GB, Wang HL, Sang LS, Wang XJ, Yang YG , Yang YG.m ⁶A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency
Cell Stem Cell, 2015,16(3):289-301.
URLPMID:25683224 [本文引用: 2]
Zhou and colleagues show that formation of m6A on mRNAs is regulated by miRNAs via a sequence pairing mechanism, and that in addition to differential distribution in pluripotent and differentiated cells, m6A has a positive influence on reprogramming to pluripotency.

[30] Perry RP, Kelley DE . Existence of methylated messenger- RNA in mouse L cells
Cell, 1974,1(1):37-42.
URL [本文引用: 1]
Messenger RNA of mouse L cells is methylated in both base and ribose moieties. On the average there are about 2.2 methyl groups per 1000 nucleotides in mRNA, a proportion which is about one-sixth that of mammalian ribosomal RNA. The variety of methylated bases in mRNA is more limited than in ribosomal RNA. A very low level of methylation is detected in heterogeneous nuclear RNA, suggesting that methylation, like polyadenylation, may constitute a post-transcriptional modification of messenger RNA precursor in eucaryotic cells.

[31] Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, Hao YJ, Ping XL, Chen YS, Wang WJ, Jin KX, Wang X, Huang CM, Fu Y, Ge XM, Song SH, Jeong HS, Yanagisawa H, Niu Y, Jia GF, Wu W, Tong WM, Okamoto A, He C, Rendtlew Danielsen JM, Wang XJ, Yang YG . FTO- dependent demethylation of N ⁶-methyladenosine regulates mRNA splicing and is required for adipogenesis
Cell Res , 2014,24(12):1403-1419.
[本文引用: 2]

[32] Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, Sun HY, Li A, Ping XL, Lai WY, Wang X, Ma HL, Huang CM, Yang Y, Huang N, Jiang GB, Wang HL, Zhou Q, Wang XJ, Zhao YL, Yang YG . Nuclear m ⁶A reader YTHDC1 regulates mRNA splicing
Mol Cell, 2016,61(4):507-519.
URLPMID:26876937 [本文引用: 2]
Xiao et02al. show that m6A reader YTHDC1 promotes exon inclusion of targeted mRNAs through facilitating SRSF3 while blocking SRSF10 mRNA binding, demonstrating how m6A reader YTHDC1 directly regulates mRNA splicing by bridging interactions oftrans- andcis-regulatory elements.

[33] Roundtree IA, Luo GZ, Zhang Z, Wang X, Zhou T, Cui Y, Sha J, Huang X, Guerrero L, Xie P, He E, Shen B , He C . YTHDC1 mediates nuclear export of N ⁶-methyladenosine methylated mRNAs
eLife , 2017, 6: pii: e31311.
[本文引用: 2]

[34] Alarcón CR, Goodarzi H, Lee H, Liu X, Tavazoie S, Tavazoie SF . HNRNPA2B1 is a mediator of m ⁶A-dependent nuclear RNA processing events
Cell, 2015,162(6):1299-1308.
URLPMID:4673968 [本文引用: 1]
The RNA-binding protein HNRNPA2B1 is a nuclear “reader” of the m6A mark, acting as an adaptor that recruits the Microprocessor complex to a subset of precursor miRNAs, facilitating their processing into mature miRNAs.

[35] Wu BX, Su SC, Patil DP, Liu HH, Gan JH, Jaffrey SR, Ma JB . Molecular basis for the specific and multivariant recognitions of RNA substrates by human hnRNP A2/B1
Nat Commun, 2018,9:420.
URL [本文引用: 1]
Human hnRNP A2/B1 is an RNA-binding protein that plays important roles in many biological processes, including maturation, transport, and metabolism of mRNA, and gene regulation of long noncoding RNAs. hnRNP A2/B1 was reported to control the microRNAs sorting to exosomes and promote primary microRNA processing as a potential m6A “reader.” hnRNP A2/B1 contains two RNA recognition motifs that provide sequence-specific recognition of RNA substrates. Here, we determine crystal structures of tandem RRM domains of hnRNP A2/B1 in complex with various RNA substrates, elucidating specific recognitions of AGG and UAG motifs by RRM1 and RRM2 domains, respectively. Further structural and biochemical results demonstrate multivariant binding modes for sequence-diversified RNA substrates, supporting a RNA matchmaker mechanism in hnRNP A2/B1 function. Moreover, our studies in combination with bioinformatic analysis suggest that hnRNP A2/B1 may mediate effects of m6A through a “m6A switch” mechanism, instead of acting as a direct “reader” of m6A modification. RNA-binding protein hnRNP A2/B1 is suggested to promote miRNA processing as a m6A 'reader'. Here, the authors determine crystal structures of RRM domains of hnRNP A2/B1 in complex with various RNA substrates and determine that hnRNP A2/B1 may function as an auxiliary factor in 'm6A switch' instead.

[36] Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T . N ⁶-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions
Nature , 2015,518(7540):560-564.
URLPMID:25719671 [本文引用: 2]
Abstract RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

[37] Liu N, Zhou KI, Parisien M, Dai Q, Diatchenko L, Pan T . N ⁶-methyladenosine alters RNA structure to regulate binding of a low-complexity protein
Nucleic Acids Res , 2017,45(10):6051-6063.
URLPMID:28334903 [本文引用: 2]
N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA), and affects almost every stage of the mRNA life cycle. The YTH-domain proteins can specifically recognize m6A modification to control mRNA maturation, translation and decay. m6A can also alter RNA structures to affect RNA–protein interactions in cells. Here, we show that m6A increases the accessibility of its surrounding RNA sequence to bind heterogeneous nuclear ribonucleoprotein G (HNRNPG). Furthermore, HNRNPG binds m6A-methylated RNAs through its C-terminal low-complexity region, which self-assembles into large particlesin vitro. The Arg-Gly-Gly repeats within the low-complexity region are required for binding to the RNA motif exposed by m6A methylation. We identified 13,191 m6A sites in the transcriptome that regulate RNA–HNRNPG interaction and thereby alter the expression and alternative splicing pattern of target mRNAs. Low-complexity regions are pervasive among mRNA binding proteins. Our results show that m6A-dependent RNA structural alterations can promote direct binding of m6A-modified RNAs to low-complexity regions in RNA binding proteins.

[38] Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C . N ⁶-methyladenosine-dependent regulation of messenger RNA stability
Nature , 2014,505(7481):117-120.
URLPMID:24284625 [本文引用: 2]
Abstract N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

[39] Du H, Zhao Y, He JQ, Zhang Y, Xi HR, Liu MF, Ma JB, Wu LG . YTHDF2 destabilizes m ⁶A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex
Nat Commun, 2016,7:12626.
URLPMID:27558897 [本文引用: 1]
Methylation at the N6 position of adenosine (m(6)A) is the most abundant RNA modification within protein-coding and long noncoding RNAs in eukaryotes and is a reversible process with important biological functions. YT521-B homology domain family (YTHDF) proteins are the readers of m(6)A, the binding of which results in the alteration of the translation efficiency and stability of m(6)A-containing RNAs. However, the mechanism by which YTHDF proteins cause the degradation of m(6)A-containing RNAs is poorly understood. Here we report that m(6)A-containing RNAs exhibit accelerated deadenylation that is mediated by the CCR4-NOT deadenylase complex. We further show that YTHDF2 recruits the CCR4-NOT complex through a direct interaction between the YTHDF2 N-terminal region and the SH domain of the CNOT1 subunit, and that this recruitment is essential for the deadenylation of m(6)A-containing RNAs by CAF1 and CCR4. Therefore, we have uncovered the mechanism of YTHDF2-mediated degradation of m(6)A-containing RNAs in mammalian cells.

[40] Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, Weng X, Chen K, Shi H, He C . N ⁶-methyladenosine modulates messenger RNA translation efficiency
Cell , 2015,161(6):1388-1399.
URLPMID:26046440 [本文引用: 2]
Human YTHDF1 binds m6A-modified mRNAs and through interactions with initiation factors and ribosomes increases translational output from those messages.

[41] Li A, Chen YS, Ping XL, Yang X, Xiao W, Yang Y, Sun HY, Zhu Q, Baidya P, Wang X, Bhattarai DP, Zhao YL, Sun BF, Yang YG . Cytoplasmic m ⁶A reader YTHDF3 promotes mRNA translation
Cell Res, 2017,27(3):444-447.
URL [本文引用: 1]

[42] Shi H, Wang X, Lu Z, Zhao BS, Ma H, Hsu PJ, Liu C, He C . YTHDF3 facilitates translation and decay of N ⁶-methyladenosine-modified RNA
Cell Res , 2017,27(3):315-328.
[本文引用: 1]

[43] Hsu PJ, Zhu Y, Ma H, Guo Y, Shi X, Liu Y, Qi M, Lu Z, Shi H, Wang J, Cheng Y, Luo G, Dai Q, Liu M, Guo X, Sha J, Shen B, He C . Ythdc2 is an N ⁶-methyladenosine binding protein that regulates mammalian spermatogenesis
Cell Res , 2017,27(9):1115-1127.
[本文引用: 3]

[44] Bailey AS, Batista PJ, Gold RS, Chen YG, de Rooij DG, Chang HY , Fuller MT . The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline
. eLife , 2017, 6, pii: e26116.
URLPMID:29087293 [本文引用: 1]
10.7554/eLife.26116.001The switch from mitosis to meiosis is the key event marking onset of differentiation in the germline stem cell lineage. In Drosophila, the translational repressor Bgcn is required for spermatogonia to stop mitosis and transition to meiotic prophase and the spermatocyte state. Here we show that the mammalian Bgcn homolog YTHDC2 facilitates a clean switch from mitosis to meiosis in mouse germ cells, revealing a conserved role for YTHDC2 in this critical cell fate transition. YTHDC2-deficient male germ cells enter meiosis but have a mixed identity, maintaining expression of Cyclin A2 and failing to properly express many meiotic markers. Instead of continuing through meiotic prophase, the cells attempt an abnormal mitotic-like division and die. YTHDC2 binds multiple transcripts including Ccna2 and other mitotic transcripts, binds specific piRNA precursors, and interacts with RNA granule components, suggesting that proper progression of germ cells through meiosis is licensed by YTHDC2 through post-transcriptional regulation.

[45] Wojtas MN, Pandey RR, Mendel M, Homolka D, Sachidanandam R , Pillai RS.Regulation of m ⁶A transcripts by the 3°→5° RNA helicase YTHDC2 is essential for a successful meiotic program in the mammalian germline
Mol Cell, 2017, 68(2): 374- 387. e12.
URLPMID:29033321 [本文引用: 2]
N 6 -methyladenosine (m 6 A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m 6 A marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m 6 A reader that is essential for male and female fertility in mice. High-throughput mapping of the m 6 A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m 6 A-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 300→500 RNA helicase activity. Furthermore, YTHDC2 recruits the 500→300 exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of m 6 A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.

[46] Jain D, Puno MR, Meydan C, Lailler N, Mason CE, Lima CD, Anderson KV , Keeney S.ketu mutant mice uncover an essential meiotic function for the ancient RNA helicase YTHDC2
. eLife, 2018, 7, pii: e30919.
URLPMID:29360036 [本文引用: 1]
10.7554/eLife.30919.001Mechanisms regulating mammalian meiotic progression are poorly understood. Here we identify mouse YTHDC2 as a critical component. A screen yielded a sterile mutant, ???ketu???, caused by a Ythdc2 missense mutation. Mutant germ cells enter meiosis but proceed prematurely to aberrant metaphase and apoptosis, and display defects in transitioning from spermatogonial to meiotic gene expression programs. ketu phenocopies mutants lacking MEIOC, a YTHDC2 partner. Consistent with roles in post-transcriptional regulation, YTHDC2 is cytoplasmic, has 3??????5??? RNA helicase activity in vitro, and has similarity within its YTH domain to an N6-methyladenosine recognition pocket. Orthologs are present throughout metazoans, but are diverged in nematodes and, more dramatically, Drosophilidae, where Bgcn is descended from a Ythdc2 gene duplication. We also uncover similarity between MEIOC and Bam, a Bgcn partner unique to schizophoran flies. We propose that regulation of gene expression by YTHDC2-MEIOC is an evolutionarily ancient strategy for controlling the germline transition into meiosis.

[47] Schwartz S, Agarwala SD, Mumbach MR, Jovanovic M, Mertins P, Shishkin A, Tabach Y, Mikkelsen TS, Satija R, Ruvkun G, Carr SA, Lander ES, Fink GR, Regev A . High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis
Cell, 2013,155(6):1409-1421.
URLPMID:3956118 [本文引用: 1]
A nearly single-base-pair resolution map of m6A mRNA methylation in yeast provides insight into the temporal dynamics of this modification during meiosis and the cis and trans components that influence the reading and writing of the mark.

[48] Zhang S, Zhao BS, Zhou A, Lin K, Zheng S, Lu Z, Chen Y, Sulman EP, Xie K, Bogler O, Majumder S, He C , Huang S. m ⁶A demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation program
Cancer Cell, 2017, 31(4): 591- 606. e6.
URLPMID:28344040 [本文引用: 1]
The dynamic and reversible N 6 -methyladenosine (m 6 A) RNA modification installed and erased by N 6 -methyltransferases and demethylases regulates gene expression and cell fate. We show that the m 6 A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs. Integrated transcriptome and m 6 A-seq analyses revealed altered expression of certain ALKBH5 target genes, including the transcription factor FOXM1. ALKBH5 demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Furthermore, a long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a critical function for ALKBH5 and provides insight into critical roles of m 6 A methylation in glioblastoma.

[49] Hosono Y, Niknafs YS, Prensner JR, Iyer MK, Dhanasekaran SM, Mehra R, Pitchiaya S, Tien J, Escara-Wilke J, Poliakov A, Chu SC, Saleh S, Sankar K, Su F, Guo S, Qiao Y, Freier SM, Bui HH, Cao X, Malik R, Johnson TM, Beer DG, Feng FY, Zhou W , Chinnaiyan AM.Oncogenic role of THOR, a conserved cancer/testis long non-coding RNA
Cell, 2017, 171(7): 1559- 1572. e20.
URLPMID:29245011 [本文引用: 1]
Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), whichexhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection.

[50] Edupuganti RR, Geiger S, Lindeboom RGH, Shi H, Hsu PJ, Lu Z, Wang SY, Baltissen MPA, Jansen P, Rossa M, Muller M, Stunnenberg HG, He C, Carell T, Vermeulen M . N ⁶-methyladenosine (m ⁶A) recruits and repels proteins to regulate mRNA homeostasis
Nat Struct Mol Biol , 2017,24(10):870-878.
URLPMID:28869609 [本文引用: 1]
RNA modifications are integral to the regulation of RNA metabolism. One abundant mRNA modification is N(6)-methyladenosine (m(6)A), which affects various aspects of RNA metabolism, including splicing, translation and degradation. Current knowledge about the proteins recruited to m(6)A to carry out these molecular processes is still limited. Here we describe comprehensive and systematic mass-spectrometry-based screening of m(6)A interactors in various cell types and sequence contexts. Among the main findings, we identified G3BP1 as a protein that is repelled by m(6)A and positively regulates mRNA stability in an m(6)A-regulated manner. Furthermore, we identified FMR1 as a sequence-context-dependent m(6)A reader, thus revealing a connection between an mRNA modification and an autism spectrum disorder. Collectively, our data represent a rich resource and shed further light on the complex interplay among m(6)A, m(6)A interactors and mRNA homeostasis.

[51] Arguello AE DeLiberto AN, Kleiner RE. RNA chemical proteomics reveals the N ⁶-methyladenosine (m ⁶A)-regulated protein-RNA interactome
J Am Chem Soc , 2017,139(48):17249-17252.
[本文引用: 2]

[52] Huang H, Weng H, Sun W, Qin X, Shi H, Wu H, Zhao BS, Mesquita A, Liu C, Yuan CL, Hu YC, Huttelmaier S, Skibbe JR, Su R, Deng X, Dong L, Sun M, Li C, Nachtergaele S, Wang Y, Hu C, Ferchen K, Greis KD, Jiang X, Wei M, Qu L, Guan JL, He C, Yang J, Chen J . Recognition of RNAN ⁶-methyladenosine by IGF2BP proteins enhances mRNA stability and translation
Nat Cell Biol , 2018,20(3):285-295.
[本文引用: 3]

[53] Fustin JM , Doi M,Yamaguchi Y,Hida H,Nishimura S,Yoshida M,Isagawa T,Morioka MS,Kakeya H,Manabe I,Okamura H. RNA-methylation-dependent RNA processing controls the speed of the circadian clock
Cell#, 2013, 155(4): 793-806.
URLPMID:24209618 [本文引用: 1]
Methylation of adenosine nucleotides (m6A), a common mRNA modification whose physiological role is not well understood, is essential to clock function. Its inhibition prolongs the circadian period due to effects on the processing of clock RNAs.

[54] Xiang Y, Laurent B, Hsu CH, Nachtergaele S, Lu Z, Sheng W, Xu C, Chen H, Ouyang J, Wang S, Ling D, Hsu PH, Zou L, Jambhekar A, He C, Shi Y . RNA m ⁶A methylation regulates the ultraviolet-induced DNA damage response
Nature, 2017,543(7646):573-576.
URLPMID:28297716
Abstract Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m 6 A) in RNA is rapidly (within 2090009min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A) + transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m 6 A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase 0202 (Pol 0202), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol 0202 overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m 6 A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m 6 A RNA serves as a beacon for the selective, rapid recruitment of Pol 0202 to damage sites to facilitate repair and cell survival.

[55] Aguilo F, Zhang F, Sancho A, Fidalgo M, Di Cecilia S, Vashisht A, Lee DF, Chen CH, Rengasamy M, Andino B, Jahouh F, Roman A, Krig SR, Wang R, Zhang W, Wohlschlegel JA, Wang J, Walsh MJ . Coordination of m ⁶A mRNA methylation and gene transcription by ZFP217 regulates pluripotency and reprogramming
Cell Stem Cell, 2015,17(6):689-704.
URLPMID:26526723
Aguillo et al. show that ZFP217 coordinates epigenetic and epitranscriptomic networks to control embryonic stem cell pluripotency and somatic cell reprogramming. ZFP217 directly activates core stem cell genes and restrains m6A deposition in their transcripts through interaction with METTL3, coupling transcription and mRNA methylation to control stemness.

[56] Geula S, Moshitch-Moshkovitz S, Dominissini D, Mansour AA, Kol N, Salmon-Divon M, Hershkovitz V, Peer E, Mor N, Manor YS, Ben-Haim MS, Eyal E, Yunger S, Pinto Y, Jaitin DA, Viukov S, Rais Y, Krupalnik V, Chomsky E, Zerbib M, Maza I, Rechavi Y, Massarwa R, Hanna S, Amit I, Levanon EY, Amariglio N, Stern- Ginossar N, Novershtern N, Rechavi G, Hanna JH . Stem cells. m ⁶A mRNA methylation facilitates resolution of naive pluripotency toward differentiation
Science, 2015,347(6225):1002-1006.
URL [本文引用: 1]

[57] Zhao BS, Wang X, Beadell AV, Lu ZK, Shi HL, Kuuspalu A, Ho RK , Yang YG.m ⁶A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition
Nature, 2017,542(7642):475-478.
URLPMID:5323276
The maternal-to-zygotic transition (MZT) is one of the most profound and tightly orchestrated processes during the early life of embryos, yet factors that shape the temporal pattern of vertebrate MZT are largely unknown. Here we show that over one-third of zebrafish maternal messenger RNAs (mRNAs) can be N6-methyladenosine (m6A) modified, and the clearance of these maternal mRNAs is facilitated by an m6A-binding protein, Ythdf2. Removal of Ythdf2 in zebrafish embryos decelerates the decay of m6A-modified maternal mRNAs and impedes zygotic genome activation. These embryos fail to initiate timely MZT, undergo cell-cycle pause, and remain developmentally delayed throughout larval life. Our study reveals m6A-dependent RNA decay as a previously unidentified maternally driven mechanism that regulates maternal mRNA clearance during zebrafish MZT, highlighting the critical role of m6A mRNA methylation in transcriptome switching and animal development.

[58] Haussmann IU, Bodi Z, Sanchez-Moran E, Mongan NP, Archer N, Fray RG , Yang YG.m ⁶A potentiates
Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature, 2016,540(7632):301-304.


[59] Lence T, Akhtar J, Bayer M, Schmid K, Spindler L, Ho CH, Kreim N, Andrade-Navarro MA, Poeck B, Helm M , Yang YG.m ⁶A modulates neuronal functions and sex determination in
Drosophila. Nature, 2016,540(7632):242-247.
[本文引用: 2]

[60] Shen L, Liang Z, Gu X, Chen Y, Teo ZW, Hou X, Cai WM, Dedon PC, Liu L, Yu H . N ⁶-methyladenosine RNA modification regulates shoot stem cell fate in Arabidopsis
Dev Cell , 2016,38(2):186-200.
[本文引用: 1]

[61] Zhang CX, Chen YS, Sun BF, Wang L, Yang Y, Ma DY, Lv JH, Heng J, Ding YY, Xue YY, Lu XY, Xiao W, Yang YG , Yang YG.m ⁶A modulates haematopoietic stem and progenitor cell specification
Nature, 2017,549(7671):273-276.
URLPMID:28869969 [本文引用: 1]
N(6)-methyladenosine (m(6)A) has been identified as the most abundant modification on eukaryote messenger RNA (mRNA). Although the rapid development of high-throughput sequencing technologies has enabled insight into the biological functions of m(6)A modification, the function of m(6)A during vertebrate embryogenesis remains poorly understood. Here we show that m(6)A determines cell fate during the endothelial-to-haematopoietic transition (EHT) to specify the earliest haematopoietic stem/progenitor cells (HSPCs) during zebrafish embryogenesis. m(6)A-specific methylated RNA immunoprecipitation combined with high-throughput sequencing (MeRIP-seq) and m(6)A individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (miCLIP-seq) analyses reveal conserved features on zebrafish m(6)A methylome and preferential distribution of m(6)A peaks near the stop codon with a consensus RRACH motif. In mettl3-deficient embryos, levels of m(6)A are significantly decreased and emergence of HSPCs is blocked. Mechanistically, we identify that the delayed YTHDF2-mediated mRNA decay of the arterial endothelial genes notch1a and rhoca contributes to this deleterious effect. The continuous activation of Notch signalling in arterial endothelial cells of mettl3-deficient embryos blocks EHT, thereby repressing the generation of the earliest HSPCs. Furthermore, knockdown of Mettl3 in mice confers a similar phenotype. Collectively, our findings demonstrate the critical function of m(6)A modification in the fate determination of HSPCs during vertebrate embryogenesis.

[62] Lv JH, Zhang YF, Gao SW, Zhang CX, Chen YS, Li W, Yang YG, Zhou Q, Liu F . Endothelial-specific m ⁶A modulates mouse hematopoietic stem and progenitor cell development via Notch signaling
Cell Res, 2018,28(2):249-252.
URLPMID:29148543 [本文引用: 1]
Endothelial-specific m6A modulates mouse hematopoietic stem and progenitor cell development via Notch signaling

[63] Xu K, Yang Y, Feng GH, Sun BF, Chen JQ, Li YF, Chen YS, Zhang XX, Wang CX, Jiang LY, Liu C, Zhang ZY, Wang XJ, Zhou Q, Yang YG, Li W . Mettl3-mediated m ⁶A regulates spermatogonial differentiation and meiosis initiation
Cell Res, 2017,27(9):1100-1114.
URLPMID:28809392 [本文引用: 1]
Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.

[64] Lin Z, Hsu PJ, Xing X, Fang J, Lu Z, Zou Q, Zhang KJ, Zhang X, Zhou Y, Zhang T, Zhang Y, Song W, Jia G, Yang X, He C, Tong MH . Mettl3-/Mettl14-mediated mRNA N ⁶-methyladenosine modulates murine spermatogenesis
Cell Res , 2017,27(10):1216-1230.
[本文引用: 1]

[65] Li L, Zang L, Zhang F, Chen J, Shen H, Shu L, Liang F, Feng C, Chen D, Tao H, Xu T, Li Z, Kang Y, Wu H, Tang L, Zhang P, Jin P, Shu Q, Li X . Fat mass and obesity- associated (FTO) protein regulates adult neurogenesis
Hum Mol Genet, 2017,26(13):2398-2411.
URL [本文引用: 1]

[66] Weng YL, Wang X, An R, Cassin J, Vissers C, Liu Y, Liu Y, Xu T, Wang X, Wong SZH, Joseph J, Dore LC, Dong Q, Zheng W, Jin P, Wu H, Shen B, Zhuang X, He C, Liu K, Song H , Ming GL.Epitranscriptomic m ⁶A regulation of axon regeneration in the adult mammalian nervous system
Neuron , 2018, 97(2): 313- 325. e6.
URLPMID:29346752 [本文引用: 1]
SummaryN6-methyladenosine (m6A) affects multiple aspects of mRNA metabolism and regulates developmental transitions by promoting mRNA decay. Little is known about the role of m6A in the adult mammalian nervous system. Here we report that sciatic nerve lesion elevates levels of m6A-tagged transcripts encoding many regeneration-associated genes and protein translation machinery components in the adult mouse dorsal root ganglion (DRG). Single-base resolution m6A-CLIP mapping further reveals a dynamic m6A landscape in the adult DRG upon injury. Loss of either m6A methyltransferase complex component Mettl14 or m6A-binding protein Ythdf1 globally attenuates injury-induced protein translation in adult DRGs and reduces functional axon regeneration in the peripheral nervous system in vivo. Furthermore, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult central nervous system is attenuated upon Mettl14 knockdown. Our study reveals a critical epitranscriptomic mechanism in promoting injury-induced protein synthesis and axon regeneration in the adult mammalian nervous system.

[67] Yoon KJ, Ringeling FR, Vissers C, Jacob F, Pokrass M, Jimenez-Cyrus D, Su Y, Kim NS, Zhu Y, Zheng L, Kim S, Wang X, Doré LC, Jin P, Regot S, Zhuang X, Canzar S, He C, Ming GL , Song H.Temporal control of mammalian cortical neurogenesis by m ⁶A methylation
Cell , 2017, 171(4): 877- 889. e17.
URLPMID:28965759 [本文引用: 1]
Abstract N 6 -methyladenosine (m 6 A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m 6 A regulates mammalian brain development is unknown. Here, we show that m 6 A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m 6 A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m 6 A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m 6 A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m 6 A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m 6 A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m 6 A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis. Copyright 2017 Elsevier Inc. All rights reserved.

[68] Li M, Zhao X, Wang W, Shi H, Pan Q, Lu Z, Perez SP, Suganthan R, He C, Bjoras M, Klungland A . Ythdf2-mediated m ⁶A mRNA clearance modulates neural development in mice
Genome Biol, 2018,19(1):69.
URL [本文引用: 1]
N6-methyladenosine (m6A) modification in mRNAs was recently shown to be dynamically regulated, indicating a pivotal role in multiple developmental processes. Most recently, it was shown that the Mettl3-Mettl14 writer complex of this mark is required for the temporal control of cortical neurogenesis. The m6A reader protein Ythdf2 promotes mRNA degradation by recognizing m6A and recruiting the mRNA decay machinery. We show that the conditional depletion of the m6A reader protein Ythdf2 in mice causes lethality at late embryonic developmental stages, with embryos characterized by compromised neural development. We demonstrate that neural stem/progenitor cell (NSPC) self-renewal and spatiotemporal generation of neurons and other cell types are severely impacted by the loss of Ythdf2 in embryonic neocortex. Combining in vivo and in vitro assays, we show that the proliferation and differentiation capabilities of NSPCs decrease significantly inYthdf261/61embryos. TheYthdf261/61neurons are unable to produce normally functioning neurites, leading to failure in recovery upon reactive oxygen species stimulation. Consistently, expression of genes enriched in neural development pathways is significantly disturbed. Detailed analysis of the m6A-methylomes ofYthdf261/61NSPCs identifies that the JAK-STAT cascade inhibitory genes contribute to neuroprotection and neurite outgrowths show increased expression and m6A enrichment. In agreement with the function of Ythdf2, delayed degradation of neuron differentiation-related m6A-containing mRNAs is seen inYthdf261/61NSPCs. We show that the m6A reader protein Ythdf2 modulates neural development by promoting m6A-dependent degradation of neural development-related mRNA targets. The online version of this article (10.1186/s13059-018-1436-y) contains supplementary material, which is available to authorized users.

[69] Ma C, Chang M, Lv H, Zhang ZW, Zhang W, He X, Wu G, Zhao S, Zhang Y, Wang D, Teng X, Liu C, Li Q, Klungland A, Niu Y, Song S, Tong WM . RNA m ⁶A methylation participates in regulation of postnatal development of the mouse cerebellum
Genome Biol, 2018,19(1):68.
URL [本文引用: 1]
N6-methyladenosine (m6A) is an important epitranscriptomic mark with high abundance in the brain. Recently, it has been found to be involved in the regulation of memory formation and mammalian cortical neurogenesis. However, while it is now established that m6A methylation occurs in a spatially restricted manner, its functions in specific brain regions still await elucidation. We identify widespread and dynamic RNA m6A methylation in the developing mouse cerebellum and further uncover distinct features of continuous and temporal-specific m6A methylation across the four postnatal developmental processes. Temporal-specific m6A peaks from P7 to P60 exhibit remarkable changes in their distribution patterns along the mRNA transcripts. We also show spatiotemporal-specific expression of m6A writers METTL3, METTL14, and WTAP and erasers ALKBH5 and FTO in the mouse cerebellum. Ectopic expression of METTL3 mediated by lentivirus infection leads to disorganized structure of both Purkinje and glial cells. In addition, under hypobaric hypoxia exposure,Alkbh5-deletion causes abnormal cell proliferation and differentiation in the cerebellum through disturbing the balance of RNA m6A methylation in different cell fate determination genes. Notably, nuclear export of the hypermethylated RNAs is enhanced in the cerebellum ofAlkbh5-deficient mice exposed to hypobaric hypoxia. Together, our findings provide strong evidence that RNA m6A methylation is controlled in a precise spatiotemporal manner and participates in the regulation of postnatal development of the mouse cerebellum. The online version of this article (10.1186/s13059-018-1435-z) contains supplementary material, which is available to authorized users.

[70] Wang CX, Cui GS, Liu XY, Xu K, Wang M, Zhang XX, Jiang LY, Li A, Yang Y, Lai WY, Sun BF, Jiang GB, Wang HL, Tong WM, Li W, Wang XJ, Yang YG, Zhou Q . METTL3-mediated m ⁶A modification is required for cerebellar development
Plos Biol, 2018,16(6):e2004880.
URL [本文引用: 2]
Author summary N6-methyladenosine (m6A) is an abundant modification in mRNA molecules and regulates mRNA metabolism and various biological processes, such as cell fate control, early embryonic development, sex determination, and diseases like diabetes and obesity. Adenosine methylation is regulated by a large methyltransferase complex and by demethylases, as well as by other binding proteins. METTL3 is one of the core subunits of the methyltransferase complex catalyzing m6A formation. However, the role of METTL3-mediated m6A in mammalian brain development remains unclear mainly because of the lack of specific spatiotemporal knockout animal models, as conventional METTL3 knockout in mice leads to early embryonic death. In this study, we specifically inactivated METTL3 in the developing mouse brain. We detected a drastic depletion of m6A accompanied by severe developmental defects in the cerebellum of these mice. Further analysis established that METTL3-mediated m6A participates in cerebellar development by controlling mRNA stability of genes related to cerebellar development and apoptosis and by regulating alternative splicing of pre-mRNAs of synapse-associated genes.

[71] Ke S, Alemu EA, Mertens C, Gantman EC, Fak JJ, Mele A, Haripal B, Zucker-Scharff I, Moore MJ, Park CY, Vågbø CB, Kusśnierczyk A, Klungland A, Darnell JE , Yang YG.rnell RB. A majority of m ⁶A residues are in the last exons, allowing the potential for 3' UTR regulation
Genes Dev, 2015,29(19):2037-2053.
URLPMID:26404942 [本文引用: 1]
Abstract We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice. 2015 Ke et al.; Published by Cold Spring Harbor Laboratory Press.

[72] Molinie B, Wang JK, Lim KS, Hillebrand R, Lu ZX, Van Wittenberghe N, Howard BD, Daneshvar K, Mullen AC, Dedon P, Xing Y , Yang YG.. m ⁶A-LAIC-seq reveals the census and complexity of the m ⁶A epitranscriptome
Nat Methods, 2016,13(8):692-698.
URLPMID:27376769 [本文引用: 1]
Abstract N(6)-Methyladenosine (m(6)A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m(6)A modified ('m(6)A levels') or the relationship of m(6)A modification(s) to alternative RNA isoforms. To deconvolute the m(6)A epitranscriptome, we developed m(6)A-level and isoform-characterization sequencing (m(6)A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m(6)A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3' untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m(6)A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m(6)A modifications.

[73] Zhou J, Wan J, Gao X, Zhang X, Jaffrey SR, Qian SB . Dynamic m ⁶A mRNA methylation directs translational control of heat shock response
Nature, 2015,526(7574):591-594.
URLPMID:26458103 [本文引用: 1]
The most abundant mRNA post-transcriptional modification isN6-methyladenosine (m6A) that has broad roles in RNA biology1-5. In mammalian cells, the asymmetric distribution of m6A along mRNAs leaves relatively less methylation in the 5′ untranslated region (5′UTR) compared to other regions6,7. However, whether and how 5′UTR methylation is regulated is poorly understood. Despite the crucial role of the 5′UTR in translation initiation, very little is known whether m6A modification influences mRNA translation. Here we show that in response to heat shock stress, m6A is preferentially deposited to the 5′UTR of newly transcribed mRNAs. We found that the dynamic 5′UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well characterized m6A “reader”. Upon heat shock stress, the nuclear YTHDF2 preserves 5′UTR methylation of stress-induced transcripts by limiting the m6A “eraser” FTO from demethylation. Remarkably, the increased 5′UTR methylation in the form of m6A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single site m6A modification in the 5′UTR enables translation initiation independent of the 5′ end m7G cap. The elucidation of the dynamic feature of 5′UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m6A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.

[74] Cui XA, Liang Z, Shen LS, Zhang Q, Bao SJ, Geng YK, Zhang B, Leo V, Vardy LA, Lu TG, Gu XF, Yu H . 5-Methylcytosine RNA methylation in Arabidopsis Thaliana
Mol Plant, 2017,10(11):1387-1399.
[本文引用: 1]

[75] Aschenbrenner J, Werner S, Marchand V, Adam M, Motorin Y, Helm M, Marx A . Engineering of a DNA polymerase for direct m ⁶A sequencing
Angew Chem Int Ed Engl, 2017,57(2):417-421.
URLPMID:29115744 [本文引用: 1]
Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics.N6‐methyladenosine (m6A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody‐based enrichment of m6A‐containing RNA prior to sequencing, since m6A modifications are generally “erased” during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6A sequencing. Therefore, we developed a screen to evolve an RT‐active KlenTaq DNA polymerase variant that sets a mark forN6‐methylation. We identified a mutant that exhibits increased misincorporation opposite m6A compared to unmodified A. Application of the generated DNA polymerase in next‐generation sequencing allowed the identification of m6A sites directly from the sequencing data of untreated RNA samples.

[76] Garalde DR, Snell EA, Jachimowicz D, Sipos B, Lloyd JH, Bruce M, Pantic N, Admassu T, James P, Warland A, Jordan M, Ciccone J, Serra S, Keenan J, Martin S McNeill L, Wallace EJ, Jayasinghe L, Wright C, Blasco J, Young S, Brocklebank D, Juul S, Clarke J, Heron AJ, Turner DJ. Highly parallel direct RNA sequencing on an array of nanopores
Nat Methods, 2018,15(3):201-206.
URLPMID:29334379 [本文引用: 1]
Abstract Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

[77] Li X, Xiong X, Wang K, Shu X, Ma S, Yi C . Transcriptome-wide mapping reveals reversible and dynamicN ¹-methyladenosine methylome
Nat Chem Biol , 2016,12(5):311-316.
[本文引用: 1]

[78] Dominissini D, Nachtergaele S, Moshitch-Moshkovitz S, Peer E, Kol N, Ben-Haim MS, Dai Q, Di Segni A, Salmon-Divon M, Clark WC, Zheng G, Pan T, Solomon O, Eyal E, Hershkovitz V, Han D, Doré LC, Amariglio N, Rechavi G, He C . The dynamic N ¹-methyladenosine methylome in eukaryotic messenger RNA
Nature , 2016,530(7591):441-446.
[本文引用: 1]

[79] Li X, Xiong X, Zhang M, Wang K, Chen Y, Zhou J, Mao Y, Lv J, Yi D, Chen XW, Wang C, Qian SB, Yi C . Base-resolution mapping reveals distinct m ¹A methylome in nuclear - and mitochondrial-encoded transcripts
Mol Cell , 2017, 68(5): 993- 1005. e9.
URLPMID:29107537 [本文引用: 1]
Gene expression can be post-transcriptionally regulated via dynamic and reversible RNA modifications. N1-methyladenosine (m1A) is a recently identified mRNA modification; however, little is known about its precise location, regulation and function. Here, we develop a base-resolution m1A profiling method, based on m1A-induced misincorporation during reverse transcription, and report distinct classes of m1A methylome in the human transcriptome. m1A in 5'-UTR, particularly those at the first nucleotide of mRNA, associate with increased translation efficiency. A different subset of m1A exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome and are dependent on the methyltransferase TRMT6/61A. Additionally, we show for the first time that m1A is prevalent in the mitochondrial-encoded transcripts. Manipulation of m1A level via TRMT61B, a mitochondria-localizing m1A methyltransferase, demonstrates that m1A in mitochondrial mRNA interferes with translation. Collectively, our approaches reveal distinct classes of m1A methylome and provide a resource for functional studies of m1A-mediated epitranscriptomic regulation.