Article Online

Articles Online (Volume 21, Issue 4)

Preface

RNA Modifications and Epitranscriptomics

Chengqi Yi, Jianhua Yang

no abstract

Page 675-677

Database

m6A-TSHub: Unveiling the Context-specific m6A Methylation and m6A-affecting Mutations in 23 Human Tissues

Bowen Song, Daiyun Huang, Yuxin Zhang, Zhen Wei, Jionglong Su, João Pedro de Magalhães, Daniel J. Rigden, Jia Meng, Kunqi Chen

As the most pervasive epigenetic marker present on mRNAs and long non-coding RNAs (lncRNAs), N6-methyladenosine (m6A) RNA methylation has been shown to participate in essential biological processes. Recent studies have revealed the distinct patterns of m6A methylome across human tissues, and a major challenge remains in elucidating the tissue-specific presence and circuitry of m6A methylation. We present here a comprehensive online platform, m6A-TSHub, for unveiling the context-specific m6A methylation and genetic mutations that potentially regulate m6A epigenetic mark. m6A-TSHub consists of four core components, including (1) m6A-TSDB, a comprehensive database of 184,554 functionally annotated m6A sites derived from 23 human tissues and 499,369 m6A sites from 25 tumor conditions, respectively; (2) m6A-TSFinder, a web server for high-accuracy prediction of m6A methylation sites within a specific tissue from RNA sequences, which was constructed using multi-instance deep neural networks with gated attention; (3) m6A-TSVar, a web server for assessing the impact of genetic variants on tissue-specific m6A RNA modifications; and (4) m6A-CAVar, a database of 587,983 The Cancer Genome Atlas (TCGA) cancer mutations (derived from 27 cancer types) that were predicted to affect m6A modifications in the primary tissue of cancers. The database should make a useful resource for studying the m6A methylome and the genetic factors of epitranscriptome disturbance in a specific tissue (or cancer type). m6A-TSHub is accessible at www.xjtlu.edu.cn/biologicalsciences/m6ats.

Page 678-694

Review Article

N6-methyladenosine and Its Implications in Viruses

Yafen Wang, Xiang Zhou

N6-methyladenine (m6A) is the most abundant RNA modification in mammalian messenger RNAs (mRNAs), which participates in and regulates many important biological activities, such as tissue development and stem cell differentiation. Due to an improved understanding of m6A, researchers have discovered that the biological function of m6A can be linked to many stages of mRNA metabolism and that m6A can regulate a variety of complex biological processes. In addition to its location on mammalian mRNAs, m6A has been identified on viral transcripts. m6A also plays important roles in the life cycle of many viruses and in viral replication in host cells. In this review, we briefly introduce the detection methods of m6A, the m6A-related proteins, and the functions of m6A. We also summarize the effects of m6A-related proteins on viral replication and infection. We hope that this review provides researchers with some insights for elucidating the complex mechanisms of the epitranscriptome related to viruses, and provides information for further study of the mechanisms of other modified nucleobases acting on processes such as viral replication. We also anticipate that this review can stimulate collaborative research from different fields, such as chemistry, biology, and medicine, and promote the development of antiviral drugs and vaccines.

Page 695-706

Original Research

Specific Regulation of m6A by SRSF7 Promotes the Progression of Glioblastoma

Yixian Cun, Sanqi An, Haiqing Zheng, Jing Lan, Wenfang Chen, Wanjun Luo, Chengguo Yao, Xincheng Li, Xiang Huang, Xiang Sun, Zehong Wu, Yameng Hu, Ziwen Li, Shuxia Zhang, Geyan Wu, Meisongzhu Yang, Miaoling Tang, Ruyuan Yu, Xinyi Liao, Guicheng Gao, Wei Zhao, Jinkai Wang

Serine/arginine-rich splicing factor 7 (SRSF7), a known splicing factor, has been revealed to play oncogenic roles in multiple cancers. However, the mechanisms underlying its oncogenic roles have not been well addressed. Here, based on N6-methyladenosine (m6A) co-methylation network analysis across diverse cell lines, we find that the gene expression of SRSF7 is positively correlated with glioblastoma (GBM) cell-specific m6A methylation. We then indicate that SRSF7 is a novel m6A regulator, which specifically facilitates the m6A methylation near its binding sites on the mRNAs involved in cell proliferation and migration, through recruiting the methyltransferase complex. Moreover, SRSF7 promotes the proliferation and migration of GBM cells largely dependent on the presence of the m6A methyltransferase. The two m6A sites on the mRNA for PDZ-binding kinase (PBK) are regulated by SRSF7 and partially mediate the effects of SRSF7 in GBM cells through recognition by insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Together, our discovery reveals a novel role of SRSF7 in regulating m6A and validates the presence and functional importance of temporal- and spatial-specific regulation of m6A mediated by RNA-binding proteins (RBPs).
研究问题： SRSF7在多种肿瘤中有促癌作用，有研究提示SRSF7在胶质瘤中异常高表达。然而，SRSF7在胶质瘤中发挥促癌作用的具体机制尚不明确。 研究方法： 我们利用生物信息学方法鉴定到SRSF7可能是一个潜在的m6A特异性调控因子，并利用Co-IP、m6A-seq、iCLIP-seq、RNA-seq、tethering、SELECT等方法，阐明SRSF7通过调控m6A的特异性修饰促进胶质瘤发展的机制。 主要结果1： SRSF7与m6A甲基转移酶复合物互作，招募METTL3促进它结合位点附近的m6A特异性修饰。 主要结果2： SRSF7特异性靶向和促进细胞增殖和迁移相关基因的m6A修饰。 主要结果 3： SRSF7以部分依赖于METTL3的方式促进胶质瘤细胞的增殖和迁移。 主要结果4： SRSF7通过调控PBK mRNA上m6A修饰促进胶质瘤细胞的增殖和迁移。

Page 707-728

Original Research

Transcriptome-wide Dynamics of m6A mRNA Methylation During Porcine Spermatogenesis

Zidong Liu, Xiaoxu Chen, Pengfei Zhang, Fuyuan Li, Lingkai Zhang, Xueliang Li, Tao Huang, Yi Zheng, Taiyong Yu, Tao Zhang, Wenxian Zeng, Hongzhao Lu, Yinghua Lv

Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N6-methyladenosine (m6A), an epigenetic modification prevalent in mRNAs, is implicated in the transcriptional regulation during spermatogenesis. However, the dynamics of m6A modification in non-rodent mammalian species remains unclear. Here, we systematically investigated the profile and role of m6A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m6A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m6A pattern between porcine and murine genes with spermatogenic function. We found that m6A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m6A modification. Our data show that m6A plays the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point are three genes (SETDB1, FOXO1, and FOXO3) that are crucial to the determination of the fate of SSCs. To the best of our knowledge, this study for the first time uncovers the expression profile and role of m6A during spermatogenesis in large animals and provides insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.

Page 729-741

Original Research

m6A Profile Dynamics Indicates Regulation of Oyster Development by m6A-RNA Epitranscriptomes

Lorane Le Franc, Bruno Petton, Pascal Favrel, Guillaume Rivière

The N6-methylation of RNA adenosines (N6-methyladenosine, m6A) is an important regulator of gene expression with critical implications in vertebrate and insect development. However, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq), we generated transcriptome-wide m6A-RNA methylomes covering the entire development of the oyster from oocytes to juveniles. Oyster RNA classes display specific m6A signatures, with messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) exhibiting distinct profiles and being highly methylated compared to transposable element (TE) transcripts. Epitranscriptomes are dynamic and correspond to the chronological steps of development (cleavage, gastrulation, organogenesis, and metamorphosis), with minimal mRNA and lncRNA methylation at the morula stage followed by a global increase. mRNA m6A levels are correlated with transcript levels, and shifts in methylation profiles correspond to expression kinetics. Differentially methylated transcripts cluster according to embryo-larval stages and bear the corresponding developmental functions (cell division, signal transduction, morphogenesis, and cell differentiation). The m6A level of TE transcripts is also regulated and peaks during the gastrulation. We demonstrate that m6A-RNA methylomes are dynamic and associated with gene expression regulation during oyster development. The putative epitranscriptome implication in the cleavage, maternal-to-zygotic transition, and cell differentiation in a lophotrochozoan model brings new insights into the control and evolution of developmental processes.

Page 742-755

Original Research

Characteristics of N6-methyladenosine Modification During Sexual Reproduction of Chlamydomonas reinhardtii

Ying Lv, Fei Han, Mengxia Liu, Ting Zhang, Guanshen Cui, Jiaojiao Wang, Ying Yang, Yun-Gui Yang, Wenqiang Yang

The unicellular green alga Chlamydomonas reinhardtii (hereafter Chlamydomonas) possesses both plant and animal attributes, and it is an ideal model organism for studying fundamental processes such as photosynthesis, sexual reproduction, and life cycle. N6-methyladenosine (m6A) is the most prevalent mRNA modification, and it plays important roles during sexual reproduction in animals and plants. However, the pattern and function of m6A modification during the sexual reproduction of Chlamydomonas remain unknown. Here, we performed transcriptome and methylated RNA immunoprecipitation sequencing (MeRIP-seq) analyses on six samples from different stages during sexual reproduction of the Chlamydomonas life cycle. The results show that m6A modification frequently occurs at the main motif of DRAC (D = G/A/U, R = A/G) in Chlamydomonas mRNAs. Moreover, m6A peaks in Chlamydomonas mRNAs are mainly enriched in the 3′ untranslated regions (3′UTRs) and negatively correlated with the abundance of transcripts at each stage. In particular, there is a significant negative correlation between the expression levels and the m6A levels of genes involved in the microtubule-associated pathway, indicating that m6A modification influences the sexual reproduction and the life cycle of Chlamydomonas by regulating microtubule-based movement. In summary, our findings are the first to demonstrate the distribution and the functions of m6A modification in Chlamydomonas mRNAs and provide new evolutionary insights into m6A modification in the process of sexual reproduction in other plant organisms.
研究问题： 莱茵衣藻是一种单细胞、单倍体真核绿藻，可以进行无性生殖和有性生殖，是研究生殖过程和生命周期的重要模式生物。m6A最为一种在真核生物mRNA种普遍存在的表观修饰，在动物和植物的有性生殖中具有重要的调控作。然而，目前尚不清楚m6A修饰是否参与莱茵衣藻有性生殖的调控。研究m6A在莱茵衣藻有性生殖过程中的调控机制将有助于解析m6A在生物体生殖和进化中的重要意义。 研究方法： 本研究对莱茵衣藻有性生殖过程中的关键时期进行了取样，并进行了转录组测序(RNA-seq)和基于抗体的m6A表观转录组测序（MeRIP-seq），全面解析了莱茵衣藻有性生殖过程中的m6A表观转录组图谱，并结合转录水平，分析了m6A修饰在莱茵衣藻有性生殖过程中的调控作用。 主要结果： 1. 首次解析了莱茵衣藻m6A表观转录组图谱，并分析了莱茵衣藻有性生殖过程中m6A修饰的分布特征。 2. 分析了m6A甲基化水平在莱茵衣藻有性生殖过程中的动态变化，暗示着m6A参与调控莱茵衣藻有性生殖过程。 3. 通过MeRIP-seq和RNA-seq测序结果关联分析，发现m6A修饰水平通常与相应mRNA的丰度呈负相关。 4. 基因差异表达分析结果显示，在莱茵衣藻有性生殖过程中，m6A可能通过调控微管运动相关基因和光合相关基因调控有性生殖。 5. 分析了莱茵衣藻中潜在的m6A调控蛋白。

Page 756-768

Original Research

RNA Methylome Reveals the m6A-mediated Regulation of Flavor Metabolites in Tea Leaves under Solar-withering

Chen Zhu, Shuting Zhang, Chengzhe Zhou, Caiyun Tian, Biying Shi, Kai Xu, Linjie Huang, Yun Sun, Yuling Lin, Zhongxiong Lai, Yuqiong Guo

The epitranscriptomic mark N6-methyladenosine (m6A), which is the predominant internal modification in RNA, is important for plant responses to diverse stresses. Multiple environmental stresses caused by the tea-withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, the effects of the m6A-mediated regulatory mechanism on flavor-related metabolic pathways in tea leaves remain relatively uncharacterized. We performed an integrated RNA methylome and transcriptome analysis to explore the m6A-mediated regulatory mechanism and its effects on flavonoid and terpenoid metabolism in tea (Camellia sinensis) leaves under solar-withering conditions. Dynamic changes in global m6A level in tea leaves were mainly controlled by two m6A erasers (CsALKBH4A and CsALKBH4B) during solar-withering treatments. Differentially methylated peak-associated genes following solar-withering treatments with different shading rates were assigned to terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylation can directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related transcripts and also indirectly influence the flavonoid, catechin, and theaflavin contents by triggering alternative splicing-mediated regulation. Our findings revealed a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a link between the m6A-mediated regulatory mechanism and the formation of tea flavor under solar-withering conditions.
研究问题： m6A修饰介导的调控机制是否参与调节茶叶萎凋加工过程中特征性风味代谢物的形成，进而影响最终茶叶风味品质的形成。 研究方法： 利用m6A表观转录组测序（MeRIP-seq）和转录组测序（RNA-seq），全面解析了乌龙茶不同遮荫率日光萎凋处理后的m6A表观转录组图谱，并结合转录水平，分析了m6A修饰在乌龙茶采后萎凋阶段风味品质形成中的调控机制。 主要结果： 1. 首次解析了茶叶m6A表观转录组图谱，并分析了乌龙茶采后萎凋过程中m6A修饰的分布特征。 2. m6A擦除蛋白是影响茶叶日光萎凋过程中总m6A修饰水平动态变化的主要因素。 3. CsALKBH4驱动的RNA去甲基化不仅可以通过调节萜类化合物合成途径相关转录本的稳定性和转录丰度，从而直接影响萜类化合物的含量。 4. RNA甲基化修饰通过介导选择性剪接机制间接影响类黄酮、儿茶素与茶黄素的蓄积水平。 5. 日光萎凋中适度遮荫处理有利于乌龙茶优异风味品质的形成。

Page 769-787

Original Research

Identifying RNA Modifications by Direct RNA Sequencing Reveals Complexity of Epitranscriptomic Dynamics in Rice

Feng Yu, Huanhuan Qi, Li Gao, Sen Luo, Rebecca Njeri Damaris, Yinggen Ke, Wenhua Wu, Pingfang Yang

Transcriptome analysis based on high-throughput sequencing of a cDNA library has been widely applied to functional genomic studies. However, the cDNA dependence of most RNA sequencing techniques constrains their ability to detect base modifications on RNA, which is an important element for the post-transcriptional regulation of gene expression. To comprehensively profile the N6-methyladenosine (m6A) and N5-methylcytosine (m5C) modifications on RNA, direct RNA sequencing (DRS) using the latest Oxford Nanopore Technology was applied to analyze the transcriptome of six tissues in rice. Approximately 94 million reads were generated, with an average length ranging from 619 nt to 1013 nt, and a total of 45,707 transcripts across 34,763 genes were detected. Expression profiles of transcripts at the isoform level were quantified among tissues. Transcriptome-wide mapping of m6A and m5C demonstrated that both modifications exhibited tissue-specific characteristics. The transcripts with m6A modifications tended to be modified by m5C, and the transcripts with modifications presented higher expression levels along with shorter poly(A) tails than transcripts without modifications, suggesting the complexity of gene expression regulation. Gene Ontology analysis demonstrated that m6A- and m5C-modified transcripts were involved in central metabolic pathways related to the life cycle, with modifications on the target genes selected in a tissue-specific manner. Furthermore, most modified sites were located within quantitative trait loci that control important agronomic traits, highlighting the value of cloning functional loci. The results provide new insights into the expression regulation complexity and data resource of the transcriptome and epitranscriptome, improving our understanding of the rice genome.
研究问题 RNA修饰是影响生命活动的重要表观调控方式，大部分依赖于cDNA的RNA测序技术制约了对RNA修饰的检测能力。高通量无偏的检测RNA修饰是研究中的难题之一，而测序技术的发展为RNA修饰鉴定提供了新的思路。 研究方法 作者利用基于Nanopore平台RNA直接测序技术（Direct RNA Sequencing，DRS）和基于Illumina的二代测序技术对水稻根、茎、叶、雄花、雌花和成熟种子胚共六个组织进行转录组测序，鉴定RNA修饰的转录本，挖掘RNA修饰与特异组织的关联性，解析其潜在的功能。 主要结果 1. 构建了水稻6个组织的动态转录组图谱 2. 鉴定到多个组织特异性表达的基因和转录本 3. m6A和m5C修饰位点在不同组织中表现为共有和特异性特征 4. m6A和m5C修饰与表达水平和poly(A)长度紧密相关 5. m6A和m5C修饰的转录本参与中心代谢并表现出组织特异性特征 6. 大多数m6A和m5C修饰的基因位于QTL位点内。

Page 788-804

Original Research

Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine Reprogramming During Early Human Development

Xiao Han, Jia Guo, Mengke Wang, Nan Zhang, Jie Ren, Ying Yang, Xu Chi, Yusheng Chen, Huan Yao, Yong-Liang Zhao, Yun-Gui Yang, Yingpu Sun, Jiawei Xu

After implantation, complex and highly specialized molecular events render functionally distinct organ formation, whereas how the epigenome shapes organ-specific development remains to be fully elucidated. Here, nano-hmC-Seal, RNA bisulfite sequencing (RNA-BisSeq), and RNA sequencing (RNA-Seq) were performed, and the first multilayer landscapes of DNA 5-hydroxymethylcytosine (5hmC) and RNA 5-methylcytosine (m5C) epigenomes were obtained in the heart, kidney, liver, and lung of the human foetuses at 13–28 weeks with 123 samples in total. We identified 70,091 and 503 organ- and stage-specific differentially hydroxymethylated regions (DhMRs) and m5C-modified mRNAs, respectively. The key transcription factors (TFs), T-box transcription factor 20 (TBX20), paired box 8 (PAX8), krueppel-like factor 1 (KLF1), transcription factor 21 (TCF21), and CCAAT enhancer binding protein beta (CEBPB), specifically contribute to the formation of distinct organs at different stages. Additionally, 5hmC-enriched Alu elements may participate in the regulation of expression of TF-targeted genes. Our integrated studies reveal a putative essential link between DNA modification and RNA methylation, and illustrate the epigenetic maps during human foetal organogenesis, which provide a foundation for for an in-depth understanding of the epigenetic mechanisms underlying early development and birth defects.
研究问题： 人胎儿发育过程中表观修饰DNA 5hmC和RNA m5C的动态变化规律是什么？两种修饰如何特异性调控不同器官发育过程？其如何影响转录因子靶区域调控基因表达？两层面的表观修饰是否存在潜在的协同调控关系？ 研究方法： 在本研究中，将123 个13–28 周人类胎儿的心脏、肾脏、肝脏和肺样本进行Nano-hmC-Seal、RNA-BisSeq和 RNA-Seq高通量测序和生物信息分析，比较不同器官发育过程中的DNA 5hmC和RNA m5C修饰特征并鉴定器官发育特异性修饰区域和基因。结合转录因子识别位置信息，解析表观修饰影响其靶基因表达变化和逆转座子调控的规律。通过综合分析三个组学特征，阐释胎儿不同器官发育表观调控规律。 主要结果1： 绘制了首个胎儿多器官DNA 5hmC和 RNA m5C表观修饰图谱。 主要结果2： 鉴定了 70,091 和 503 个器官发育阶段特异性差异羟甲基化区域和m5C修饰的mRNA。 主要结果3： 关键转录因子TBX20、PAX8、KLF1、TCF21和 CEBPB对不同器官的发育至关重要。 主要结果4： 富含5hmC的Alu元件可能参与调节转录因子靶向基因的表达。 数据连接： https://ngdc.cncb.ac.cn/gsa-human/browse/HRA000705

Page 805-822

Original Research

NSUN2-mediated mRNA m5C Modification Regulates the Progression of Hepatocellular Carcinoma

Dan Song, Ke An, Wenlong Zhai, Luyao Feng, Yingjie Xu, Ran Sun, Yueqin Wang, Yun-Gui Yang, Quancheng Kan, Xin Tian

RNA modifications affect many biological processes and physiological diseases. The 5-methylcytosine (m5C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m5C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that the hypermethylated genes mainly participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m5C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNAs of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 expression in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Taken together, our study demonstrates the vital role of NSUN2 in the progression of HCC.
研究问题 研究发现，RNA表观修饰影响肿瘤的发生发展。肝癌的致病因素与发病机制复杂，但RNA m5C在肝癌发生发展中扮演着怎样的角色未曾有研究报道。基于以上疑问，我们提出RNA m5C在肝癌中具有怎样的分布特征？NSUN2作为主要催化mRNA m5C修饰的酶，如何通过影响修饰水平进而调控肝癌发生发展？ 研究方法 本研究通过构建肝癌癌组织Bis-seq文库，结合生物信息学分析绘制了肝癌的mRNA m5C图谱。同时也发现了潜在引起肝癌的m5C修饰基因及相关信号通路。进一步，利用实时定量PCR和基因敲除等技术，验证NSUN2对特定基因表达和细胞信号通路的调控作用。此外，通过细胞实验评估NSUN2影响肝细胞癌细胞对索拉非尼的敏感性以及细胞周期调控功能。 主要结果 1. 绘制肝癌组织的m5C甲基化图谱。结合甲基化修饰组数据和转录组数据分析发现了肝癌中高甲基化高表达的基因主要参与磷酸激酶信号通路，如Ras和PI3K-Akt通路等。 2. NSUN2在肝细胞癌组织中高表达，并与多个高m5C修饰且高表达基因呈正相关，包括GRB2、RNF115、AATF、ADAM15、RTN3和HDGF。通过实时定量PCR验证，在肝细胞癌细胞中，NSUN2的下调导致GRB2、RNF115和AATF等mRNA表达显著降低。 3. NSUN2通过调节Ras信号通路活性，影响肝细胞癌细胞对索拉非尼的敏感性，并调控肝细胞癌的细胞周期。

Page 823-833

Original Research

Dynamic Landscapes of tRNA Transcriptomes and Translatomes in Diverse Mouse Tissues

Peng Yu, Siting Zhou, Yan Gao, Yu Liang, Wenbing Guo, Dan Ohtan Wang, Shuaiwen Ding, Shuibin Lin, Jinkai Wang, Yixian Cun

Although the function of tRNAs in the translational process is well established, it remains controversial whether tRNA abundance is tightly associated with translational efficiency (TE) in mammals. Moreover, how critically the expression of tRNAs contributes to the establishment of tissue-specific proteomes in mammals has not been well addressed. Here, we measured both tRNA expression using demethylase-tRNA sequencing (DM-tRNA-seq) and TE of mRNAs using ribosome-tagging sequencing (RiboTag-seq) in the brain, heart, and testis of mice. Remarkable variation in the expression of tRNA isodecoders was observed among different tissues. When the statistical effect of isodecoder-grouping on reducing variations is considered through permutating the anticodons, we observed an expected reduction in the variation of anticodon expression across all samples, an unexpected smaller variation of anticodon usage bias, and an unexpected larger variation of tRNA isotype expression at amino acid level. Regardless of whether or not they share the same anticodons, the isodecoders encoding the same amino acids are co-expressed across different tissues. Based on the expression of tRNAs and the TE of mRNAs, we find that the tRNA adaptation index (tAI) and TE are significantly correlated in the same tissues but not between tissues; and tRNA expression and the amino acid composition of translating peptides are positively correlated in the same tissues but not between tissues. We therefore hypothesize that the tissue-specific expression of tRNAs might be due to post-transcriptional mechanisms. This study provides a resource for tRNA and translation studies, as well as novel insights into the dynamics of tRNAs and their roles in translational regulation.
研究问题： 尽管tRNAs在翻译进程中的功能已被明确定义，但是在哺乳动物中，tRNAs的组织特异性表达及其在翻译进程中的调控机制仍然有待进一步研究。 研究方法： 我们采用基于去甲基化酶处理的tRNA测序技术（DM-tRNA-seq）并结合核糖体标记测序方法（RiboTag-seq）评估小鼠大脑、心脏和睾丸组织中tRNAs的表达以及信使核糖核酸（mRNAs）的翻译效率，并从tRNA同型解码子（tRNA isodecoder）、基于编码氨基酸水平的同型tRNA（tRNA isotype）、翻译进程中密码子使用偏好性以及肽链合成中氨基酸组成等多个层面解析tRNAs表达在翻译进程中的调控功能。 主要结果1： tRNA isodecoders在不同小鼠组织中呈现出动态表达。 主要结果2： 基于编码氨基酸水平的同型tRNA具有显著的组织特异性。 主要结果 3： 组织内tRNAs表达更好地适配高翻译效率基因的翻译需求，而不是组织之间特异的高翻译效率基因。 主要结果4： tRNAs表达与同一组织内的氨基酸组成呈正相关，但在组织之间无关联。

Page 834-849

Original Research

RNA Structural Dynamics Modulate EGFR-TKI Resistance Through Controlling YRDC Translation in NSCLC Cells

Boyang Shi, Ke An, Yueqin Wang, Yuhan Fei, Caixia Guo, Qiangfeng Cliff Zhang, Yun-Gui Yang, Xin Tian, Quancheng Kan

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) positively affect the initial control of non-small cell lung cancer (NSCLC). Rapidly acquired resistance to EGFR-TKIs is a major hurdle in successful treatment. However, the mechanisms that control the resistance of EGFR-TKIs remain largely unknown. RNA structures have widespread and crucial functions in many biological regulations; however, the functions of RNA structures in regulating cancer drug resistance remain unclear. Here, the psoralen analysis of RNA interactions and structures (PARIS) method is used to establish the higher-order RNA structure maps of EGFR-TKIs-resistant and -sensitive cells of NSCLC. Our results show that RNA structural regions are enriched in untranslated regions (UTRs) and correlate with translation efficiency (TE). Moreover, yrdC N6-threonylcarbamoyltransferase domain containing (YRDC) promotes resistance to EGFR-TKIs. RNA structure formation in YRDC 3′ UTR suppresses embryonic lethal abnormal vision-like 1 (ELAVL1) binding, leading to EGFR-TKI sensitivity by impairing YRDC translation. A potential therapeutic strategy for cancer treatment is provided using antisense oligonucleotide (ASO) to perturb the interaction between RNA and protein. Our study reveals an unprecedented mechanism through which the RNA structure switch modulates EGFR-TKI resistance by controlling YRDC mRNA translation in an ELAVL1-dependent manner.
研究问题 表皮生长因子受体酪氨酸激酶抑制剂（Epidermal growth factor receptor-tyrosine kinase inhibitor，EGFR-TKI）在非小细胞肺癌（non-small cell lung cancer，NSCLC）的临床治疗中起到了重要的作用。但EGFR-TKI肿瘤耐药的快速发生则阻碍其在临床上的广泛应用，且其耐药的机制目前还尚不清楚。在目前的研究中已经发现，RNA结构异常会导致神经退行性疾病的及心血管发育异常等疾病的发生。但关于RNA结构，尤其是转录组水平的RNA结构，对癌症的发生与耐药机制的研究尚未见报道。 研究方法 本研究从RNA二级结构入手，通过补骨脂素分析RNA相互作用及结构方法（psoralen analysis of RNA interactions and structures method, PARIS）构建非小细胞肺癌发生与耐药的RNA结构图谱。结合生物信息学分析建立非小细胞肺癌发生与耐药的RNA结构调控组，进一步通过分子细胞，生物化学等实验技术，以及转录组组和翻译组学，重点鉴定非小细胞肺癌发生与耐药过程中的RNA结构调控机制，联合RNA结构动态调控/RBP/RNA代谢调控/肿瘤发生与耐药这几个重要的生物学过程及事件，为肿瘤的发生与耐药的研究提供新的思路，也为肿瘤的临床治疗提供新的理论基础。 主要结果 1. PARIS方法构建非小细胞肺癌细胞获得性耐药模型的RNA结构组图谱，并发现RNA结构区域主要富集在非翻译区（Untranslated Regions，UTR）、翻译起始位点和终止密码子区域，表明其在转录后调节和翻译控制中的作用。 2. 在非小细胞肺癌EGFR-TKI耐药过程中，RNA翻译效率呈现动态性变化，且与RNA结构呈负相关。 3. RNA结构动态变化通过embryonic lethal abnormal vision-like 1 (ELAVL1)依赖性的机制来调控N6-threonylcarbamoyltransferase domain containing (YRDC)翻译，进而调控非小细胞肺癌的EGFR-TKI耐药调控新机制。

Page 850-865

Original Research

Multi-omics of Circular RNAs and Their Responses to Hormones in Moso Bamboo (Phyllostachys edulis)

Yongsheng Wang, Huihui Wang, Huiyuan Wang, Ruifan Zhou, Ji Wu, Zekun Zhang, Yandong Jin, Tao Li, Markus V. Kohnen, Xuqing Liu, Wentao Wei, Kai Chen, Yubang Gao, Jiazhi Ding, Hangxiao Zhang, Bo Liu, Chentao Lin, Lianfeng Gu

Circular RNAs (circRNAs) are endogenous non-coding RNAs with covalently closed structures, which have important functions in plants. However, their biogenesis, degradation, and function upon treatment with gibberellins (GAs) and auxins (1-naphthaleneacetic acid, NAA) remain unknown. Here, we systematically identified and characterized the expression patterns, evolutionary conservation, genomic features, and internal structures of circRNAs using RNase R-treated libraries from moso bamboo (Phyllostachys edulis) seedlings. Moreover, we investigated the biogenesis of circRNAs dependent on both cis- and trans-regulation. We explored the function of circRNAs, including their roles in regulating microRNA (miRNA)-related genes and modulating the alternative splicing of their linear counterparts. Importantly, we developed a customized degradome sequencing approach to detect miRNA-mediated cleavage of circRNAs. Finally, we presented a comprehensive view of the participation of circRNAs in the regulation of hormone metabolism upon treatment of bamboo seedlings with GA and NAA. Collectively, our study provides insights into the biogenesis, function, and miRNA-mediated degradation of circRNAs in moso bamboo.
研究问题： 毛竹属单子叶禾本科竹亚科中最具经济价值的笋材两用植物，中国在种质资源、种植面积及竹材产量等方面稳居世界第一。但目前毛竹环状RNA响应激素诱导的分子机制仍不清楚，同时关于激素诱导毛竹环状RNA的功能、形成和降解机制也未见报道。 研究方法 为了探索在转录水平探索环状RNA的降解信息，本研究设计了一个新的降解组文库构建流程同时检测环状RNA和线性RNA的降解信息。 主要结果： 该研究论文以毛竹为研究材料，探究了如下内容： 主要成果1： 通过RNase R消化去除线性RNA富集环状RNA方法识别了激素诱导的环状RNA。 主要成果2：系统探究了毛竹激素诱导环状RNA的形成、降解和功能。 主要成果3：选取关键调控激素诱导环状RNA进行稳定过表达，可观察到株高的变化。为后续进一步深入研究毛竹环状RNA在激素响应中的调控功能奠定了前期基础。 数据连接： 基因组测序数据以及注释文件均保存至中国国家生物信息中心国家基因组科学数据中心。可在https://ngdc.cncb.ac.cn免费下载（GSA: CRA007877）。

Page 866-885

Letter

Screening Linear and Circular RNA Transcripts from Stress Granules

Shuai Chen, Jinyang Zhang, Fangqing Zhao

Stress granules (SGs) are cytoplasmic ribonucleoprotein assemblies formed under stress conditions and are related to various biological processes and human diseases. Previous studies have reported the regulatory role of some proteins and linear RNAs in SG assembly. However, the relationship between circular RNAs (circRNAs) and SGs has not been discovered. Here, we screened both linear RNAs and circRNAs in SGs using improved total RNA sequencing of purified SG cores in mammalian cells and identified circular transcripts specifically localized in SGs. circRNAs with higher SG-related RNA-binding protein (RBP) binding abilities are more likely to be enriched in SGs. Furthermore, some SG-enriched circRNAs are differentially expressed in hepatocellular carcinoma (HCC) and adjacent tissues. These results suggest the regulatory role of circRNAs in SG formation and provide insights into the biological function of circRNAs and SGs in HCC.

Page 886-893