Volume: 16, Issue: 2

Review

Structural Insights into N6-methyladenosine (m6A) Modification in the Transcriptome

Jinbo Huang, Ping Yin

More than 100 types of chemical modifications in RNA have been well documented. Recently, several modifications, such as N6-methyladenosine (m6A), have been detected in mRNA, opening the window into the realm of epitranscriptomics. The m6A modification is the most abundant modification in mRNA and non-coding RNA (ncRNA). At the molecular level, m6A affects almost all aspects of mRNA metabolism, including splicing, translation, and stability, as well as microRNA (miRNA) maturation, playing essential roles in a range of cellular processes. The m6A modification is regulated by three classes of proteins generally referred to as the “writer” (adenosine methyltransferase), “eraser” (m6A demethylating enzyme), and “reader” (m6A-binding protein). The m6A modification is reversibly installed and removed by writers and erasers, respectively. Readers, which are members of the YT521-B homology (YTH) family proteins, selectively bind to RNA and affect its fate in an m6A-dependent manner. In this review, we summarize the structures of the functional proteins that modulate the m6A modification, and provide our insights into the m6A-mediated gene regulation.
目前在RNA中已经发现有超过100种的化学修饰。最近在真核mRNA上发现一类新修饰，即腺嘌呤甲基化（m6A），拓宽了对表观转录组学（epitranscriptomics）的认识。m6A修饰是在mRNA和非编码RNA（ncRNA）中最丰富的一种修饰。m6A影响mRNA的代谢，包括剪接、翻译和稳定性，以及microRNA的成熟。这些被m6A调节的RNA代谢过程在这一系列的细胞进程中扮演着重要角色。在体内，m6A修饰主要被三类蛋白分子调节，包括催化在RNA甲基化的甲基转移酶，一般被称为“编码器”（writer，m6A甲基转移酶）；去除在RNA上甲基化的去甲基化酶，被称为“消码器”（eraser，m6A去甲基化酶）；识别RNA上甲基化的蛋白，被称为“读码器”（reader，m6A结合蛋白）。因此，m6A甲基化修饰是个可逆的过程，分别由编码器写入和消码器消除，并且还可以通过读码器识别，参与并影响到一系列的生物学过程。在这篇综述中，我们从结构生物学的角度，系统地总结了这些蛋白或者复合物的结构特征，对调节m6A修饰的分子机理进行了阐述和讨论，为m6A介导的基因表达调控提供了一些见解。

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Review

YTH Domain: A Family of N6-methyladenosine (m6A) Readers

Shanhui Liao, Hongbin Sun, Chao Xu

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N6-methyladenosine (m6A) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, m6A can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the m6A modification is reversible and dynamic. Moreover, m6A is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the m6A recognition by YTH domain-containing proteins, which would shed new light on m6A-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.
像蛋白和DNA一样，不同的RNA分子也会进行不同的修饰。大量证据表明这些RNA修饰是一种复杂代码，用来调控RNA的行为和很多重要的生物学功能。m6A是真核生物RNA中存在的一种最为丰富的内部RNA修饰，m6A修饰在信使RNA，转运RNA，核糖体RNA和长链非编码 RNA等RNA中广泛存在。在哺乳动物细胞中，m6A可通过甲基转移酶复合体形成并被去甲基化酶去除，从而保证了这种修饰是动态可逆的。此外，m6A可被含有YT521-B同源（YTH）结构域的蛋白识别，进而指导不同的复合物调节RNA信号传导途径，如：RNA代谢，RNA剪接，RNA折叠和蛋白质翻译。因此，我们总结了近期在研究含有YTH结构域蛋白识别m6A的分子机制方面的进展，这将会为研究m6A特异性识别带来新的亮点，并为将来确定识别其他RNA修饰的蛋白提供了线索。

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Review

Applications of RNA Indexes for Precision Oncology in Breast Cancer

Liming Ma, Zirui Liang, Hui Zhou, Lianghu Qu

Precision oncology aims to offer the most appropriate treatments to cancer patients mainly based on their individual genetic information. Genomics has provided numerous valuable data on driver mutations and risk loci; however, it remains a formidable challenge to transform these data into therapeutic agents. Transcriptomics describes the multifarious expression patterns of both mRNAs and non-coding RNAs (ncRNAs), which facilitates the deciphering of genomic codes. In this review, we take breast cancer as an example to demonstrate the applications of these rich RNA resources in precision medicine exploration. These include the use of mRNA profiles in triple-negative breast cancer (TNBC) subtyping to inform corresponding candidate targeted therapies; current advancements and achievements of high-throughput RNA interference (RNAi) screening technologies in breast cancer; and microRNAs as functional signatures for defining cell identities and regulating the biological activities of breast cancer cells. We summarize the benefits of transcriptomic analyses in breast cancer management and propose that unscrambling the core signaling networks of cancer may be an important task of multiple-omic data integration for precision oncology.
肿瘤精准治疗（Precision oncology）的目标旨在依据癌症病人遗传信息的个体化差异，来为这些病人设计最合适的治疗策略。目前基因组学已经给我们提供了与驱动癌症的关键突变以及高风险位点相关的大量有用的数据。然而，如何将这些信息转化为治疗药物仍然是一个艰巨的挑战。转录组学揭示了各种信使RNA（mRNA）和非编码RNA（non-coding RNA，ncRNA）的表达情况，因而能够很好地破译基因组学中的遗传密码。在本综述中，我们以乳腺癌作为例子，重点介绍这些丰富的RNA资源在精准医疗药物开发中的应用，主要表现在3个方面：（1）利用mRNA表达谱对三阴性乳腺癌（triple-negative breast cancer，TNBC）进行分类并提出相应的候选靶向治疗策略；（2）高通量RNA干预技术（RNA interference， RNAi）在乳腺癌中的研究进展和成果；（3）微小RNA（microRNA， miRNA）是表征细胞谱系和调控乳腺癌细胞生物学活性的重要功能分子。我们总结了转录组学在乳腺癌治疗中的应用，提出在肿瘤精准治疗中不同类别的组学的数据整合的一个重要任务在于揭示隐藏其中的核心信号调控网络。

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Review

The Role of Exportin-5 in MicroRNA Biogenesis and Cancer

Ke Wu, Juan He, Wenchen Pu, Yong Peng

MicroRNAs (miRNAs) are conserved small non-coding RNAs that play an important role in the regulation of gene expression and participate in a variety of biological processes. The biogenesis of miRNAs is tightly controlled at multiple steps, such as transcription of miRNA genes, processing by Drosha and Dicer, and transportation of precursor miRNAs (pre-miRNAs) from the nucleus to the cytoplasm by exportin-5 (XPO5). Given the critical role of nuclear export of pre-miRNAs in miRNA biogenesis, any alterations of XPO5, resulting from either genetic mutation, epigenetic change, abnormal expression level or posttranslational modification, could affect miRNA expression and thus have profound effects on tumorigenesis. Importantly, XPO5 phosphorylation by ERK kinase and its cis/trans isomerization by the prolyl isomerase Pin1 impair XPO5′s nucleo-to-cytoplasmic transport ability of pre-miRNAs, leading to downregulation of mature miRNAs in hepatocellular carcinoma. In this review, we focus on how XPO5 transports pre-miRNAs in the cells and summarize the dysregulation of XPO5 in human tumors.
MicroRNA（miRNA）是一类小非编码RNA，具有调节基因表达的功能，因此在许多生物学过程中发挥重要作用。miRNA的生物合成对肿瘤的发生发展至关重要，而miRNA合成是一个多步调节过程，需要由XPO5介导miRNA前体（pre-miRNA）从细胞核转运至细胞质，进而参与调节mRNA的翻译，沉默基因的表达。这种XPO5介导的出核机制，不是序列依赖性的，能够通过其蛋白结构识别各种pre-miRNA。 研究发现，XPO5不仅能转运pre-miRNA，还可以转运某些tRNA和mRNA。因此，XPO5基因或蛋白水平的调节，可作为miRNA生物发生过程的限速步骤。 在肿瘤发生发展过程中，XPO5的失调可导致pre-miRNA核积累和成熟miRNA表达的紊乱。有多项研究证明，这种失调是由基因突变，表观遗传学的改变，以及XPO5蛋白的异常表达或翻译后修饰（PTMs）引起的。从表观遗传学上来看，XPO5启动子中CpG岛上的DNA超甲基化通常会导致XPO5的基因沉默。此外，XPO5存在两种遗传变异类型，分别为单核苷酸多态性（Single nucleotide polymorphisms ,SNPs）和拷贝数变化（copy number variations ,CNVs）。统计分析表明，XPO5相关的miRNA-SNPs与肿瘤的发展及预后有明显联系。但迄今为止，还没有直接的证据支持XPO5的CNV与任何人类疾病有关。然而，由于微卫星的不稳定，XPO5基因发生了遗传突变，导致XPO5的失活。比如，XPO5的C-末端区域是pre-miRNA / XPO5 / Ran-GTP三元复合物形成的关键之处，那么缺失C-末端的XPO5蛋白不能结合并转运pre-miRNA，从而导致pre-miRNA的核积累并减少成熟的miRNA表达。有趣的是，XPO5不仅能够调控miRNA的表达，还能被miRNA调控。研究发现，miR-138的过表达可降低XPO5的水平。近期的重要研究指出，在肝癌中的XPO5被ERK激酶磷酸化后，脯氨酰顺反异构酶Pin1改变了它的构象，破坏其转运pre-miRNA的能力，从而导致了成熟miRNAs的下调表达。这项新研究代表着XPO5的PTMs机制领域取得了重大的进展。同时，有研究指出，XPO5蛋白还涉及其他修饰，但其生物学影响还需进一步探究。 在本文中，我们将聚焦细胞中XPO5转运pre-miRNAs的生物学过程，对 XPO5的各类异常与癌症发生发展之间的关系进行了详细总结讨论。这对于研究miRNA提供了重要信息和线索。

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Original Research

Comparative Analysis of Human Genes Frequently and Occasionally Regulated by m6A Modification

Yuan Zhou, Qinghua Cui

The m6A modification has been implicated as an important epitranscriptomic marker, which plays extensive roles in the regulation of transcript stability, splicing, translation, and localization. Nevertheless, only some genes are repeatedly modified across various conditions and the principle of m6A regulation remains elusive. In this study, we performed a systems-level analysis of human genes frequently regulated by m6A modification (m6Afreq genes) and those occasionally regulated by m6A modification (m6Aocca genes). Compared to the m6Aocca genes, the m6Afreq genes exhibit gene importance-related features, such as lower dN/dS ratio, higher protein–protein interaction network degree, and reduced tissue expression specificity. Signaling network analysis indicates that the m6Afreq genes are associated with downstream components of signaling cascades, high-linked signaling adaptors, and specific network motifs like incoherent feed forward loops. Moreover, functional enrichment analysis indicates significant overlaps between the m6Afreq genes and genes involved in various layers of gene expression, such as being the microRNA targets and the regulators of RNA processing. Therefore, our findings suggest the potential interplay between m6A epitranscriptomic regulation and other gene expression regulatory machineries.
正如DNA和组蛋白修饰作为DNA水平上的表观遗传调控的核心组分一样，RNA腺嘌呤碱基上第6位N上的甲基化修饰（即m6A修饰）也是一种关键的、RNA水平上的表观遗传修饰。m6A甲基化修饰被发现能够调控转录本稳定性、可变剪接、翻译、定位等重要生物过程。然而，目前m6A调控的机制尚未完全解明。随着高通量RNA m6A甲基化测序（MeRIP-seq）数据的积累，我们现在可以通过生物信息学方法比较不同实验得到的m6A甲基化谱，挖掘其中潜在的调控规律。有意思的是，我们通过对现有人类m6A甲基化谱数据的整理发现，只有少数基因能够在多种情况下被反复修饰，而这一现象背后的原理仍不清楚。在本工作中，我们系统比较分析了在多种情况下频繁和偶尔发生m6A修饰的基因。相对于偶尔发生m6A修饰的基因而言，频繁发生m6A修饰的基因表现出一些与基因重要性有关的特征，如更低的dN/dS比率，更高的蛋白互作网络的度，更低的组织表达特异性等。这说明重要基因倾向于受到m6A修饰调控。而信号转导网络分析则提示，频繁发生m6A修饰的基因倾向于作为信号转导通路的下游组分、高度连接的信号转导接头蛋白、以及一些特殊网络模体（如非协调的前馈环）的成员出现。m6A这种信号转导网络的拓扑特征与miRNA靶基因在信号转导网络的拓扑特征有一定相似性。确实，功能富集分析进一步表明频繁发生m6A修饰的基因与microRNA靶基因、参与到RNA加工调控的基因等存在显著重叠。因此，我们的分析结果提示m6A表观转录组调控与其它基因表达调控机制存在潜在的功能关联与互动。

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Method

GoldCLIP: Gel-omitted Ligation-dependent CLIP

Jiaqi Gu, Ming Wang, Yang Yang, Ding Qiu, Yiqun Zhang, Jinbiao Ma, Yu Zhou, Gregory J. Hannon, Yang Yu

Protein–RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein–RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.

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Application Note

SPORTS1.0: A Tool for Annotating and Profiling Non-coding RNAs Optimized for rRNA- and tRNA-derived Small RNAs

Junchao Shi, Eun-A Ko, Kenton M. Sanders, Qi Chen, Tong Zhou

High-throughput RNA-seq has revolutionized the process of small RNA (sRNA) discovery, leading to a rapid expansion of sRNA categories. In addition to the previously well-characterized sRNAs such as microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and small nucleolar RNA (snoRNAs), recent emerging studies have spotlighted on tRNA-derived sRNAs (tsRNAs) and rRNA-derived sRNAs (rsRNAs) as new categories of sRNAs that bear versatile functions. Since existing software and pipelines for sRNA annotation are mostly focused on analyzing miRNAs or piRNAs, here we developed the sRNA annotation pipelineoptimized for rRNA- and tRNA-derived sRNAs (SPORTS1.0). SPORTS1.0 is optimized for analyzing tsRNAs and rsRNAs from sRNA-seq data, in addition to its capacity to annotate canonical sRNAs such as miRNAs and piRNAs. Moreover, SPORTS1.0 can predict potential RNA modification sites based on nucleotide mismatches within sRNAs. SPORTS1.0 is precompiled to annotate sRNAs for a wide range of 68 species across bacteria, yeast, plant, and animal kingdoms, while additional species for analyses could be readily expanded upon end users’ input. For demonstration, by analyzing sRNA datasets using SPORTS1.0, we reveal that distinct signatures are present in tsRNAs and rsRNAs from different mouse cell types. We also find that compared to other sRNA species, tsRNAs bear the highest mismatch rate, which is consistent with their highly modified nature. SPORTS1.0 is an open-source software and can be publically accessed at https://github.com/junchaoshi/sports1.0.
高通量RNA测序技术大大加快了发现未知RNA的过程。近年来小RNA的种类在不断扩增，除了已知的一些小RNA，例如microRNA（miRNA），Piwi-interacting RNA（piRNA）和小核仁RNA（snoRNA）外，最近一系列研究集中在两种全新的小RNA： tRNA来源的小RNA（tsRNA）以及rRNA来源的小RNA（rsRNA）。由于现有的小RNA注释工具主要集中用于分析miRNA或者piRNA，对其他新型小RNA的支持并不完善。为了同时注释多种小RNA，特别是针对tsRNA和rsRNA，本文开发了SPORTS1.0（small RNA annotation pipeline optimized for rRNA- and tRNA- derived small RNAs）作为优化的小RNA注释工具。此外，SPORTS1.0还能通过RNA序列错配预测潜在的RNA修饰位点。为了方便用户使用，SPORTS1.0提供了广达68个物种（横跨动、植物及微生物）的预编译注释数据库，同时用户也可以自定义注释所用的数据库。作为举例，本文利用SPORTS1.0分析发现，tsRNA和rsRNA存在于不同小鼠细胞类型中并各自具有独特的分布模式。此外，本文还发现相较于其他小RNA，tsRNA具有更高的错配率，这与tRNA中具有更高的RNA修饰比率相符。SPORTS1.0是一款开源软件并可下载自https://github.com/junchaoshi/sports1.0。

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Corrigendum

Corrigendum to “Pharmacogenomics of Drug Metabolizing Enzymes and Transporters: Relevance to Precision Medicine” [Genomics Proteomics Bioinformatics 14 (5) (2016) 298–313]

Shabbir Ahmed, Zhan Zhou, Jie Zhou, Shu-Qing Chen

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