华中农业大学 植物科学技术学院，湖北 武汉 430070
收稿日期：2019-12-24；接收日期：2020-03-25；网络出版时间：2020-04-15
基金项目：国家自然科学基金(No.31100268), 湖北省自然科学基金(No.2016CFB438), 高校科研基本业务费(No.2662015PY168)资助
摘要：信使RNA (Messenger RNA，mRNA)上的表观修饰对于转录本的稳定性和翻译活性有重要影响。在不同生物体的不同发育时期和不同组织器官中，特异转录本不同位点存在的核苷修饰影响mRNA的前体剪切、成熟mRNA的稳定性以及其翻译为蛋白质的效率。目前已知的170多种修饰核苷中在mRNA上发现的只占极少数，由于mRNA的丰度低、组织和发育特异性强等特点，研究mRNA特异位点的核苷修饰有很大的技术难度。近些年随着meRIP等技术的进步，mRNA核苷修饰功能的研究得到了长足的发展，特别是针对m6A、m5C等甲基化修饰的研究已经相当深入。文中简要回顾近年来mRNA核苷修饰领域的研究进展，对不同位点和不同类型的修饰核苷在不同物种生长发育中的调控作一总结，并对未来的研究热点和技术瓶颈展开讨论。
关键词：信使RNA核苷修饰发育调控表观转录组学RNA表观遗传学
Modified nucleosides in eukaryotic messenger RNAs and their roles in developmental regulation
Xue Liu, Tao Zhang, Xiaoqi Zhou, Lun Guan, Peng Chen
Faculty of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
Received: December 24, 2019; Accepted: March 25, 2020; Published: April 15, 2020
Supported by: National Natural Science Foundation of China (No.31100268), Natural Science Foundation of Hubei Province, China (No.2016CFB438), Fundamental Research Funds for the Central Universities, China (No.2662015PY168)
Corresponding author: Peng Chen. Tel: +86-27-87281399; E-mail: chenpeng@mail.hzau.edu.cn.
Abstract: Epigenetic nucleoside modifications are critical for the stability and translational efficiency of messenger RNA. Depending on the organism, developmental stage, and tissue/organ investigated, the location and abundance of these nucleoside modifications may differ, which in turn influence the splicing event, half-life time of mature mRNA, as well as translation efficiency. Among the approximately 170 RNA nucleoside modifications, only a handful are found in mRNAs. The low abundance and high organ specificity make it a challenging work to study the role of each specific mRNA nucleoside modification. However, with the technical advances in recent years, including meRIP, great progress has been achieved, especially on the function of m6A and m5C epigenetic markers in eukaryotes. This review summarizes recent progress on nucleoside modifications of messenger RNAs, on their distribution on transcripts and their role in regulating growth and development. We also discuss the technical bottleneck and key issues for future investigation.
Keywords: messenger RNAmodified nucleosidesdevelopmental regulationepitranscriptomeRNA epigenetics
信使RNA (Messenger RNA，mRNA)是遗传信息表达的核心分子，mRNA作为蛋白质合成的模板，其碱基序列决定着蛋白质装配时的氨基酸序列。中心法则中，遗传信息表达的解码过程需要转运RNA (tRNA)上的反密码子和信使RNA (mRNA)上的密码子完成配对和携带入对应的氨基酸掺入多肽链，由于修饰核苷的存在，即使是相同的核苷酸序列，也可最终表达出不同的遗传信息。已有较多的研究证明，转运RNA (tRNA)和核糖体RNA (rRNA)的加工和成熟过程涉及大量的化学修饰，在某些情况下，这些额外的基团修饰对其正常折叠和行使功能至关重要[1]。mRNA也可以像rRNA和tRNA一样被修饰。目前已经知道的RNA核苷修饰有170余种(Modomics Database，http://modomics.genesilico.pl/modifications/)，其中大部分出现在tRNA和rRNA中，mRNA中已知的修饰仅有18种，其中大多为甲基化修饰(图 1)。其中5种：1-甲基腺苷(1-methyladenosine，m1A)、假尿嘧啶(Pseudouridine，ψ)、5-羟甲基胞苷(5-hydroxymethylcytidine，hm5C)、6-甲酰腺苷(6-formyladenosine，(f6A)以及6-羟甲基腺苷(6-hydroxymethyladenosine，hm6A)发现较晚，目前还未被RNA Modification Database (https://mods.rna.albany.edu/)收录。
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| 图 1 mRNA上分布的18种修饰核苷 Fig. 1 18 modified nucleosides found on mRNA. |
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不同的修饰核苷在mRNA上的分布不同(图 2)：7-甲基鸟苷(7-methylguansoine，m7G)主要分布于mRNA的5′帽子区域，在mRNA内部也有分布[2]；6-methyladenosine (m6A)在哺乳动物中主要分布在mRNA的终止密码子附近，3′-UTR区域(3′-untranslated regions)以及长外显子内[3]，在植物mRNA的起始密码子和poly A尾巴上游也有分布[4-5]；5-methylcytidine (m5C)主要出现在mRNA的CDS (Coding sequence)区[6]；m1A在mRNA的5′-UTR (5′-untranslated regions)、CDS、3′-UTR区域都存在[7-8]；ψ主要分布在mRNA的CDS和3′-UTR区域[9]；hm5C主要分布在mRNA的CDS区域[10]。DNA与RNA的主要差别在于核糖2′-C上分别是-OH和-H，一个基团的不同却使DNA和RNA在结构和功能呈现巨大的差异，可见修饰基团的存在对RNA结构和功能的影响。由于RNA的不稳定性、结构复杂性以及检测技术有限，表观遗传学中RNA来源核苷修饰的研究比DNA上的研究进展慢。2011年第一个RNA去甲基化酶FTO (Fat-mass and obesity associated protein，m6A去甲基酶)的发现揭示了RNA修饰的可逆性[11]，是RNA表观遗传学研究的里程碑。近些年，随着meRIP等技术的快速发展，不同生物全转录组范围内的RNA修饰谱图得到揭示，越来越多的研究表明mRNA上表观修饰对生物体生长发育具有重要的调控作用。本文针对研究较多的6种mRNA上的核苷修饰，包括ψ、m7G、m1A、m6A、m5C和hm5C，从其合成、分布和功能上进行阐述和讨论。
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| 图 2 m7G、m1A、m6A、m5C、hm5C和ψ修饰核苷在mRNA上的区间分布 Fig. 2 Distribution of m7G, m1A, m6A, m5C, hm5C and ψ in mRNA. |
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1 假尿嘧啶(ψ)假尿嘧啶是尿苷(1-核糖尿苷)的异构体(5-核糖尿苷)，被认为是第5种核苷酸。假尿嘧啶是非编码RNA (Non-coding RNA)中最丰富的一种修饰[12-14]，在tRNA和rRNA中也存在，具有稳定RNA结构的功能[15-17]。mRNA上的假尿嘧啶影响mRNA的剪切[13]。在酵母中改变rRNA的假尿嘧啶修饰可以影响其对于抗生素的敏感性[18-19]。在哺乳动物中，假尿嘧啶修饰与先天性胰岛功能不良、核糖体合成紊乱以及癌症的发生相关[20]。利用pseudo-seq、ψ-seq、PSI-seq和CeU-Seq技术可实现人或酵母mRNA上单碱基分辨率下ψ的位点鉴定，数据表明哺乳动物中ψ/U的相对丰度约为0.2%–0.4%[21]。
mRNA的假尿嘧啶修饰由位点特异的snoRNA引导的PUSs (假尿嘧啶合成酶)催化形成，人类细胞中有23个蛋白含有PUS结构域[22]，合成的ψ主要富集在mRNA的CDS和3′-UTR区域[9, 21, 23-24]。mRNA的假尿嘧啶修饰有3个主要功能：1)改变密码子；2)影响转录本稳定性；3)应激反应应答[23, 25-26]。在酵母中尿嘧啶(U)被替换为假尿嘧啶(ψ)之后，原本的无义密码便可改为编码氨基酸。当酵母受到热激刺激时，由PUS7介导的假尿嘧啶位点突增；反之，PUS7缺失时含有这些ψ位点的mRNA水平下降[23]。在刚地弓形虫中的研究发现，TgPUS1突变体中ψ位点比野生型寄生虫中更稳定[27]，由此推测mRNA的假尿嘧啶修饰可能具有双向作用，在不同的生物体、基因或者不同条件下可能会增强mRNA的稳定性或者降低其稳定性。人类细胞在热激或者是H2O2处理下mRNA上的假尿嘧啶修饰水平升高，而在饥饿刺激下则会下降；酵母在营养不足的情况下和人细胞处于血清饥饿的情况下，mRNA上的假尿嘧啶化修饰水平都发生变化，可见mRNA上的大多数假尿嘧啶修饰与细胞对环境信号的应答有关[9]。
2 m7Gm7G甲基化是指RNA分子鸟嘌呤第7位氮原子上的甲基化修饰。m7G是目前发现的唯一一个在真核生物mRNA的5′端帽子结构区域出现的核苷修饰[2]。mRNA的5′帽子结构对于mRNA具有重要作用，5′帽子结构可以促使mRNA与核糖体的结合，m7Gppp结构使mRNA形成封闭的5′端，可有效防止mRNA的降解，此外5′帽子结构还影响前体mRNA的剪切、3′末端的多聚腺苷酸化以及mRNA的出核运输[28-32]。
1975年就有****发现真核生物mRNA 5′帽子结构区域的m7G修饰促进蛋白质的合成，且消除m7G修饰后相对应的mRNA就不能正常翻译[33]。1976年在卤虫藻胚胎中发现，帽子绑定蛋白(Cap-binding protein)可识别mRNA 5′端的m7GpppN的结构，促进mRNA与核糖体的结合，进而影响翻译进程[34]。后来发现，m7G不仅仅存在于mRNA的5′端帽子结构区域，在mRNA的内部同样存在。真核生物mRNA内部m7G含量为0.4–5.3/105，5′帽子结构区域m7G含量为1.0–4.9/104。植物mRNA的内部m7G含量明显高于哺乳动物。此外，研究还发现真核生物mRNA中的m7G修饰可以被动态调控。经过镉处理，水稻中m7G去帽基因表达量上升，mRNA 5′端帽子结构和内部的m7G水平均降低，内部m7G含量在水稻不同发育阶段变化趋势相同，说明mRNA内部m7G相对稳定，而5′端帽子结构的m7G与环境胁迫的应答相关[2]。
3 m6Am6A甲基化是指RNA分子腺嘌呤第6位氮原子上的甲基化修饰。20世纪70年代，在哺乳动物和植物中首次发现了mRNA上m6A的修饰，之后在病毒、果蝇、酵母等物种中陆续发现了m6A的存在[35-40]。已有研究表明，m6A修饰和mRNA的稳定性、剪接加工、翻译以及microRNA的加工有关，影响干细胞命运、生物节律等生物过程。随着m6A-seq、MeRIP、miCLIP等新技术的产生，m6A在全转录组范围内的分布越来越清晰。2012年Dominissini等利用m6A-seq技术发现人类细胞中mRNA和长链非编码RNA (Long non- coding RNAs)上有超过10 000个m6A修饰位点[3]，Meyer等利用MeRIP-seq技术发现7 676个哺乳类基因的mRNA上有m6A的修饰[41]。这些研究还表明mRNA上的m6A位点在人和鼠之间高度保守，主要富集在终止密码子附近、3′-UTR区域、长外显子内以及可变剪切位点内。在植物材料中，Bodi等发现拟南芥转录组中m6A修饰位点主要位于3′ poly A尾巴上游的100–150 nt的范围内[4]。Li等利用MeRIP-seq技术鉴定到水稻愈伤组织和叶片中8 138个和14 253个转录本上含有m6A修饰，并且修饰位点倾向分布于翻译的起始位点和终止位点附近[5]。与哺乳动物m6A保守基序“GRACH” (R=A/G；H=A/U/C)有区别的是，水稻愈伤组织中m6A保守基序为“RAGRAG”，而叶片中m6A保守基序为“UGUAMM” (M=C/A)[5]。水稻花序中m6A的保守基序与水稻叶片中的保守基序相似，为“UGWAMH” (W=U/A)[42]。拟南芥中m6A的保守基序与水稻也不同，为“RRACH”[43]。由此可见在不同物种、不同发育阶段以及不同组织中m6A位点的合成和识别可能存在高度的物种和组织特异性。
m6A是目前所知的唯一由合成蛋白(m6A writer)、去除蛋白(m6A eraser)和识别蛋白(m6A reader)构成的甲基组系统。近年来有多篇综述报道了mRNA上m6A修饰强大的调控功能[44-46]，下面简要介绍下影响m6A合成及分布的这3类蛋白。
3.1 合成蛋白(m6A writer)在哺乳动物中，m6A甲基转移酶复合体由甲基转移酶类3 (methyltransferase-like3，METTL3)、甲基转移酶类14 (METTL14)、Wilm肿瘤关联蛋白(Wilm’s tumor 1-associating protein，WTAP)、KIAA1429/VIRMA、HAKAI、RNA结合蛋白15 (RNA Binding Motif Protein15，RBM15)和锌指蛋白C3H结构域蛋白13 (Zinc finger CCCH domain- containing protein 13，ZC3H13)组成。WTAP、METTL3和METTL14主要富集在核小点处，METTL3是甲基转移酶复合体的催化中心，METTL14序列上与METTL3相似，但是METTL14不具有独立的体外甲基化酶活性，主要负责活化METTL3和招募RNA与METTL3反应[47-49]。WTAP是甲基化酶的构架蛋白，主要起稳定METTL类蛋白之间的互作，既可影响转录，也可影响RNA的剪切[50]。KIAA1429和RBM15主要起识别甲基化靶向位点的作用。拟南芥中发现HAKAI蛋白调控m6A的合成，动物细胞中HAKAI突变也造成m6A的合成受阻[51]。ZC3H13通过与WTAP、VIR和HAKAI形成蛋白复合体，将整个m6A合成复合体锚定于细胞核内[52-53]。
多个研究发现m6A writer的突变可导致不同的表型甚至胚胎致死(表 1)。例如酿酒酵母，只有在减数分裂时期的mRNA才具有m6A修饰，IME4/METTL3的缺失影响酵母的出芽和减数分裂过程[54-55]。酵母中Kar4/METTL14的突变造成单倍体配子融合失败[56-57]。斑马鱼中METTL3和WTAP的表达富集于脑部，基因敲除导致胚胎细胞凋亡增加，斑马鱼头部和脑部发育缺陷，生育能力下降[58]。果蝇的METTL3和METTL14突变体在传代能力、飞行技能、神经发育和性别决定等方面表现出不同程度的缺陷[59-64]。果蝇的m6A标记影响XIST和Sxl等母本mRNA的特异选择和剪切，从而影响性别的决定[65]。小鼠METTL3、WTAP或RBM15的缺失导致胚胎干细胞失去分化能力并导致胚胎致死[50, 66-68]；METTL14的突变影响精细胞发育和个体的育性[69]。人类细胞中METTL3缺失抑制胚胎干细胞分化，造成生物钟周期延长，METTL3可以促进癌症细胞中蛋白质的翻译，从而影响多种肿瘤疾病进程[70-72]。拟南芥MTA (METTL3的同系物)突变体和VIR突变体都是在胚胎的球形期停止发育，其中MTA突变体中m6A水平减少了将近90%，突变体花器官异常、无顶端优势，生长模式发生改变[4, 41]。拟南芥FIP37 (WTAP的同系物)突变体m6A水平减少约85%，顶端分生组织过度繁殖，植株生长不正常[73]。拟南芥hakai突变体中m6A水平相对野生型降低35%，但没有明显的表型[51]。因此，拟南芥中除了HAKAI，其他m6A甲基转移酶复合体成员缺失对于胚胎发育都至关重要，但是各组分对于植物发育的影响强度又各有不同。
表 1 mRNA常见修饰核苷的分布和功能Table 1 Distribution and function of modified nucleosides commonly found on mRNA
| Modification | Distribution | Abundance | Proteins involved | Protein classification | Biological roles | Reference |
| ψ | CDS 3′-UTR | 0.2%–0.4% | PUSs | Synthetase | Affect the sensitivity of yeast to antibiotics; Related to congenital islet dysfunction, ribosome synthesis disorder and cancer in mammal | [23, 25-26] |
| m7G | 5′Cap | 0.01%–0.05% | RNMT | Synthetase | Affects the synthesis of proteins; Responses to environmental stress | [2, 33-34] |
| eIF4E, CBP20/CBP80 | Binding protein | |||||
| m6A | CDS 3′-UTR | 0.4%–1.50% | METTL3, METTL14, WTAP, KIAA1429, HAKAI, RBM15, ZC3H13 | m6A methyltransferase | Affect the budding and meiosis of yeast; Affecting head and brain development and fertility of zebrafish; Affects the reproduction, flight skill, neurodevelopment and sex determination of fruit fly; Inhibiting differentiation of embryonic stem cells and prolonging the biological clock cycle of human; Abnormal organs, no apical dominance and changed growth pattern in Arabidopsis | [4, 41, 54-55, 58-64] |
| FTO, ALKBH5 | m6A demethylase | |||||
| YTH | m6A reader | |||||
| m1A | 5′-UTR CDS 3′-UTR | 0.015%–0.16% | TRMT6/TRMT61A, TRMT61B, TRMT10C | Synthetase | Regulates translation stability and protein synthesis | [100, 145] |
| ALKBH3 | m1A demethylase | |||||
| m5C | CDS 3′-UTR | ~0.43% | TRM4, NSUN2 | Methyltransferase | Affecting the body size development and embryonic stem cell differentiation in mouse and human; Affects memory in fruit fly and human; Affects body size, retina, liver and brain development of zebrafish; Affects root development and oxidative stress in Arabidopsis | [123-129] |
| Tet | Demethylase | |||||
| hm5C | CDS | 0.000 1%– 0.000 44% | Tet | Demethylase | Affects the brain development in human | [10, 133] |
表选项
3.2 去除蛋白(m6A eraser)目前已经发现的m6A eraser包括FTO和ALKBH5，它们都属于ALKBH家族，可以去除包括DNA和mRNA上的m6A修饰[11, 74-76]。最早的ALKBH蛋白是大肠杆菌中鉴定出来的，它通过氧化反应去除双链DNA上的m1A甲基基团，参与修复DNA烷基化损伤[74]。人类细胞中ALKBH家族蛋白有9个，包括ALKBH 1-8和FTO。在大鼠中，ALKBH5在睾丸中表达量最高，其突变会影响精母细胞的减数分裂和大鼠育性[76-77]。早期研究显示FTO与人类肥胖疾病相关，且影响人体内多巴胺的水平，FTO缺失的小鼠脂肪组织减少，产后生长缓慢[78]。在其他动物细胞中的研究发现FTO与细胞分化及肿瘤的形成有关[75, 79-82]。FTO主要定位于细胞核中，降低FTO的表达引起RNA上m6A水平的升高[11, 83]。拟南芥中没有发现FTO同源蛋白，但是有13个ALKBH蛋白。ALKBH家族蛋白种类多、分布广，有可能识别单链或双链DNA、单链RNA或双链RNA，也有可能作为蛋白上的去甲基酶，不仅可以去除m6A，还有可能去除其他种类的甲基化修饰。ALKBH9B和ALKBH10B被证实有体外去甲基化的功能，ALKBH10B具有体内去甲基化活性[84]。被AMV侵染的拟南芥中ALKBH9B活性降低，病毒胞内复制减慢，推测可能和ALKBH9B与病毒粒子结合后去除了病毒RNA上的m6A有关[85]。拟南芥alkbh10突变体营养生长受到抑制，开花延迟[84]。与ALKBH类蛋白类似，FTO蛋白不仅能去除m6A和m6Am，还能识别除mRNA外的其他底物，关于m6A eraser还有很多问题尚待研究。
3.3 识别蛋白(m6A reader)如上文所述，m6A修饰水平的变化可引起生物体各种表型，可见m6A修饰对于生物的生长发育的重要性。细胞体内m6A修饰的变化必须通过m6A reader，即m6A识别蛋白来发挥作用。目前发现的m6A reader主要是哺乳动物中的YTH蛋白家族。YTH是一类保守的蛋白家族，在人类、小鼠、酵母、果蝇、水稻、拟南芥中都存在，主要分为2个亚类：YTHDF和YTHDC。人类细胞的YTHDF2是第一个被鉴定的YTH蛋白，定位于细胞质，该蛋白包含2个功能域：C端含YTH结构域直接结合m6A；N端可使mRNA重新定位于P小体，促使其降解[86]。YTHDC1属于细胞核定位的m6A识别蛋白，可以与pre-mRNA剪切因子SRSF3相互作用，抑制剪切因子SRSF10与mRNA的结合，影响mRNA的剪切，从而影响小鼠卵细胞的发育[87]。酿酒酵母的Mrb1属于YTH蛋白家族；而Mmi1则是裂殖酵母中发现的YTH蛋白，调控酵母的生殖生长[88]。拟南芥中含有13个YTH类蛋白，其中ECT2、ECT3或ECT4的突变造成叶片表皮毛发育异常[89-90]。2018年Huang等利用RNA pull down及CLIP-seq等技术发现，人类细胞中的IGF2BP3也可结合含有GG(m6A)C基序的mRNA，这种结合增强mRNA的稳定性，利于其在胞内储存[91]。
4 m1Am1A甲基化是指RNA分子腺嘌呤第1位氮原子上的甲基化修饰。m1A是RNA修饰中比较重要且常见的一种修饰。m1A修饰不仅使腺嘌呤多了一个甲基，还使其在生理条件下附了一个正电荷，影响了RNA的结构及与蛋白的互作。单个电荷的不同可以使蛋白和DNA之间的亲和力相差100–1 000倍[92]。关于m1A修饰对tRNA结构和功能的影响已有报道[93-95]，有研究显示tRNA上的m1A修饰与环境胁迫相关[96-97]，而rRNA上的m1A修饰影响核糖体的合成和细菌对抗生素的耐受性[98-99]。
由于mRNA在胞内丰度低，修饰检测困难，因此mRNA上的修饰研究相对缓慢。在酵母、小鼠和人类等上千个转录组中都检测到m1A的存在。TRMT6/TRMT61A复合体负责tRNA上m1A58 (第58位)的甲基化，也可催化mRNA上m1A的形成，但需要与tRNA类似的基序GUUCRA和T-loop结构的存在[100]。哺乳动物中m1A/A的相对丰度为0.015%–0.16%[7]，从分布上看，m1A在mRNA的5′-UTR、CDS、3′-UTR都存在，主要富集于起始密码子附近及5′-UTR的GC富集区[7]。m1A修饰在不同物种中相对保守，由于其位于起始密码子附近，因此可以促进翻译效率[7-8]。在碱性条件下，m1A可以发生化学重排，转换成m6A[101]。人类细胞中发现mRNA上的m1A修饰可以被ALKBH3去除[102-103]。m1A核苷修饰与翻译的稳定性及蛋白的合成相关，在不同的生理条件下(如热激、H2O2、饥饿状态)，m1A核苷修饰水平是动态变化的，但是其变化背后的调控机制尚不清楚。
5 m5Cm5C甲基化是指RNA分子胞嘧啶第5位氮原子上的甲基化修饰。早期研究发现DNA上的m5C修饰对于转录沉默和基因组印记具有重要作用[104-105]。RNA上m5C修饰的研究主要集中在tRNA和rRNA[6, 104, 106-107]，mRNA和lncRNA上的m5C修饰报道较晚[108-109]。tRNA中的m5C主要集中在可变环和反密码子环，对于维持tRNA的二级结构、密码子的识别、tRNA的代谢以及对于氧化胁迫的感应具有重要作用[102, 104, 110-114]。m5C修饰影响rRNA的加工、结构，有研究报道rRNA上的m5C修饰与生物体寿命相关[115]。此外，还有研究发现m5C修饰可以维持ncRNA的稳定性[108]。
在HeLa细胞的2 243种RNA上发现5 399个m5C的修饰位点，其中94% (5 063/5 399)的位点出现在mRNA上[6]。m5C位点主要集中在CDS区域(约占总数的45%)，其中55%分布在CG富集区域，28%分布在CHG富集区域，17%分布在CHH富集区域(H=A/C/U)[6]。在小鼠组织的3 904个mRNA中共发现9 788个m5C的修饰位点，小鼠组织mRNA的平均m5C水平达20.6%–23.2%，与人类HeLa细胞含量类似。小鼠中m5C位点分布与人类HeLa细胞相似，主要集中于CG富集区和CDS紧邻翻译起始位点下游区域[6, 116]。mRNA上的m5C核苷修饰与ALYREF结合，介导mRNA的出核运动[6]。拟南芥mRNA中的m5C位点主要集中在3′-UTR[117]，预示着和动物中不同的调控模式。
在真核生物中有2种甲基转移酶，催化mRNA和其他非编码RNA上的m5C修饰：一种是TRDMT1，即已知的DNA甲基转移酶(DNMT2)，参与动物、植物和裂殖酵母的tRNA修饰[107, 118-120]；另一种是TRM4 (酵母)或NSUN2 (动物)[113, 121-122]。NSUN2与哺乳动物癌细胞增殖，干细胞的自我更新及分化相关，nsun2–/–的小鼠雄性不育、体型变小、表皮分化缺陷，胚胎干细胞的自我更新和分化受影响[123-124]；人类研究中发现，NSUN2缺失的病人智力缺陷，体格小[125-128]。其他物种的研究发现，NSUN2突变的果蝇短期记忆能力缺陷[125]，而斑马鱼缺失DNMT2酶活性时，个体体型变小，胚胎的视网膜、肝脏和大脑发育缺陷[129]。
植物中m5C的相关报道很少。在拟南芥发现有TRM4A和TRM4B两个同源基因[130-131]，其中trm4b突变体中mRNA的m5C修饰水平下降，根比较短，对于氧化胁迫更敏感，tRNA稳定性降低[125]。
6 hm5Chm5C是m5C的胞嘧啶第5位甲基发生氧化反应，将一个H变为OH后所得产物。该种核苷修饰于1978年首次从小麦幼苗的rRNA中发现[132]，但是直到最近人们才在mRNA中检测到hm5C核苷修饰的存在[133]。2014年有****发现Tet不仅可以使DNA形成hm5C修饰，也可催化RNA产生该种核苷修饰[133]。利用MeRIP-seq技术在果蝇中进行的研究发现，RNA中的hm5C修饰主要分布在CDS区，且在UC含量高的区域富集[10]。通过GO分析发现，mRNA上的hm5C修饰位点在与胚胎发育相关的基因中较丰富[10]，推测mRNA上的hm5C核苷修饰可能参与调控胚胎发育。翻译活性较高的mRNA含有较多的hm5C修饰[10]，由此推测hm5C修饰可能参与调控基因的表达。缺少Tet的果蝇，RNA的羟甲基化水平下降，且果蝇的大脑发育受损[10]。但是由于检测技术的限制，我们对于hm5C修饰的合成细节和调控模式还了解甚少，在植物中也未有关于mRNA的hm5C修饰的报道。
7 mRNA核苷修饰的检测技术随着科技的进步，mRNA核苷修饰的检测技术也在快速发展。早期mRNA修饰常用放射性标记技术[134-136]和薄层色谱法[137-138]来分析，但是这些方法成本高，操作过程繁琐，且无法用于大规模的检测和修饰位点的准确定位。质谱技术(LC-MS/MS，Liquid chromatography-tandem mass spectrometry)和抗体免疫印迹法(Dot-blotting)可实现修饰核苷的定量分析[11, 76]，但是同样也不能定位修饰位点。免疫沉淀法(Immunoprecipitation，IP)与二代测序技术(Next generation sequencing，NGS)结合形成IP-seq技术, 应用较多的有ChIP-seq[139]、PAR-CLIP[140-141]和MeRIP-seq[41]，前两种技术用于检测DNA/RNA与蛋白的结合，不能做定量分析，对于修饰位点的定位停留在数十个碱基到数百个碱基的区段，MeRIP-seq可以通过查找保守基序来确定修饰位点，但是该方法分析的是总RNA水平上的核苷修饰水平，且此方法的精确度受数据分析方法、序列比对软件和测序深度等因素的影响较大。与MeRIP-seq相类似的另一种m6A修饰水平检测方法m6A-seq[3]可检测mRNA上的碱基修饰，分辨率在200 nt左右。miCLIP (m6A individual-nucleotide-resolution cross-linking and immunoprecipitation)[142]、SCARLET[143]、PA-m6A-seq (Photo-crosslinking- assisted m6A sequencing strategy)[144]等技术可达单核苷酸的分辨率，局限性包括：miCLIP对抗体的依赖性高；SCARLET无法实现高通量；PA-m6A-seq需要引入4-硫代尿苷(4-thiouridine, 4SU)，适用于动物细胞实验。
8 总结与展望近些年，RNA表观遗传学得到了越来越广泛的关注，新的技术手段的出现大大加速了RNA修饰的研究进展。但是目前仍有部分RNA修饰缺少直接有效的检测手段，且在转录本层面的检测精度有待提高。现有的170余种RNA修饰中，mRNA修饰仅占18种，当前的研究主要聚集在修饰位点的鉴定和修饰缺陷的表型分析。未来更大的挑战是追踪这些修饰位点的动态变化，以及从深层次揭示修饰的改变如何影响基因/蛋白的表达和细胞的生命过程。
mRNA上部分修饰核苷，例如m6A和m5C的合成和去除，是动态可逆的，这种动态调控预示着在基因表达调控中的巨大潜能。哺乳动物中关于mRNA核苷修饰对mRNA加工、稳定性、翻译过程的影响，以及如何影响细胞分化、胚胎发育、应激反应、癌症发展和病毒感染等，已有很多研究成果，但植物中的相关研究还很有限，特别是大宗农作物中几乎未见报道。未来在植物中的工作将有助于我们对mRNA表观修饰对真核生物的生长发育调控有更全面的理解。
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