对孢子化卵囊和未孢子卵囊mRNA m6A修饰的比较分析为柔嫩艾美耳球虫的发育生物学提供了新见解

doi:10.1016/j.jia.2023.07.011

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (1): 239-250.DOI: 10.1016/j.jia.2023.07.011

对孢子化卵囊和未孢子卵囊mRNA m⁶A修饰的比较分析为柔嫩艾美耳球虫的发育生物学提供了新见解

收稿日期:2023-03-27 修回日期:2023-05-26 出版日期:2024-01-20 发布日期:2024-01-06

New insights into developmental biology of Eimeria tenella revealed by comparative analysis of mRNA N6-methyladenosine modification between unsporulated oocysts and sporulated oocysts

Qing Liu¹, Bingjin Mu¹, Yijing Meng¹, Linmei Yu¹, Zirui Wang¹, Tao Jia¹, Wenbin Zheng¹, Wenwei Gao¹, Shichen Xie^{1, 2#}, Xingquan Zhu^1#

¹Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China
²Research Center for Parasites & Vectors, College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China

Received:2023-03-27 Revised:2023-05-26 Online:2024-01-20 Published:2024-01-06
About author:Qing Liu, E-mail: lqsxau@163.com; #Correspondence Shichen Xie, Tel: +86-354-6286886, E-mail: xieshichen221@163.com; Xingquan Zhu, Tel: +86-354-6286886, E-mail: xingquanzhu1@hotmail.com
Supported by:

This work was supported by the National Natural Science Foundation of China (31902298), the Shanxi Provincial Key Research and Development Program, China (2022ZDYF126), the Fund for Shanxi “1331 Project”, China (20211331-13), the Science and Technology Innovation Program of Shanxi Agricultural University, China (2017YJ10), and the Special Research Fund of Shanxi Agricultural University for High-level Talents, China (2021XG001).

摘要/Abstract

摘要：

相关研究表明，N6-腺苷酸甲基化（N6-methyladenosine, m⁶A）修饰在影响RNA命运方面发挥重要作用，并且与许多物种的细胞生长和发育过程密切相关，但目前尚无关于柔嫩艾美耳球虫m⁶A修饰的报道。为了解析mRNA上的m⁶A修饰在柔嫩艾美耳球虫生长发育过程中的作用，本研究分别利用m⁶A MeRIP测序、RNA测序和4D-label free定量蛋白质组学技术，检测了柔嫩艾美耳球虫孢子化卵囊和未孢子化卵囊mRNA上的m⁶A修饰情况、差异mRNA和差异蛋白，并进行了生物信息学分析。m⁶A MeRIP测序结果表明，m⁶A修饰在CDS区中最丰富，其次是终止密码子。与未孢子卵囊相比，在孢子化卵囊中检测到3903个高甲基化基因和3178个低甲基化的基因。对差异甲基化的基因和差异mRNA进行关联分析的结果表明，大多数基因的m⁶A修饰与mRNA丰度呈正相关。我们随机选取了4个基因进行荧光定量PCR和m⁶A MeRIP-PCR验证，该结果和m⁶A MeRIP测序及RNA测序数据一致。GO和KEGG注释分析显示，这些差异甲基化且mRNA丰度存在差异的基因与regulation of gene expression、epigenetic、microtubule、autophagy-other和TOR signaling等相关。此外，我们将存在差异甲基化但在mRNA丰度上没有差异的基因与蛋白组学数据进行关联分析，结果表明，一共有96个差异甲基化的基因在mRNA丰度上没有差异但在蛋白丰度上存在显著差异。GO和KEGG注释分析显示，这96个基因可能参与虫体的细胞生物合成和代谢。我们首次绘制了柔嫩艾美耳球虫孢子化卵囊和未孢子卵囊转录组范围的m⁶A修饰图谱，通过与RNA测序数据和蛋白质组学数据的联合分析，揭示了m⁶A修饰在虫体发育过程中的潜在调控作用，并发现m⁶A修饰可能通过不依赖于mRNA水平的机制影响虫体发育过程中mRNA的翻译。

Abstract:

Evidence showed that N6-methyladenosine (m⁶A) modification plays a pivotal role in influencing RNA fate and is strongly associated with cell growth and developmental processes in many species. However, no information regarding m⁶A modification in Eimeria tenella is currently available. In the present study, we surveyed the transcriptome-wide prevalence of m⁶A in sporulated oocysts and unsporulated oocysts of E. tenella. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) analysis showed that m⁶A modification was most abundant in the coding sequences, followed by stop codon. There were 3,903 hypermethylated and 3,178 hypomethylated mRNAs in sporulated oocysts compared with unsporulated oocysts. Further joint analysis suggested that m⁶A modification of the majority of genes was positively correlated with mRNA expression. The mRNA relative expression and m⁶A level of the selected genes were confirmed by quantitative reverse transcription PCR (RT-qPCR) and MeRIP-qPCR. GO and KEGG analysis indicated that differentially m⁶A methylated genes (DMMGs) with significant differences in mRNA expression were closely related to processes such as regulation of gene expression, epigenetic, microtubule, autophagy-other and TOR signaling. Moreover, a total of 96 DMMGs without significant differences in mRNA expression showed significant differences at protein level. GO and pathway enrichment analysis of the 96 genes showed that RNA methylation may be involved in cell biosynthesis and metabolism of E. tenella. We firstly present a map of RNA m⁶A modification in E. tenella, which provides significant insights into developmental biology of E. tenella.

Key words: Eimeria tenella , , m⁶A , RNA methylation , MeRIP-seq , RNA-seq , proteomic analysis

对孢子化卵囊和未孢子卵囊mRNA m⁶A修饰的比较分析为柔嫩艾美耳球虫的发育生物学提供了新见解 [J]. Journal of Integrative Agriculture, 2024, 23(1): 239-250.

Qing Liu, Bingjin M, Yijing Meng, Linmei Yu, Zirui Wang, Tao Jia, Wenbin Zheng, Wenwei Gao, Shichen Xie, Xingquan Zhu.

New insights into developmental biology of Eimeria tenella revealed by comparative analysis of mRNA N6-methyladenosine modification between unsporulated oocysts and sporulated oocysts [J]. Journal of Integrative Agriculture, 2024, 23(1): 239-250.

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对孢子化卵囊和未孢子卵囊mRNA m⁶A修饰的比较分析为柔嫩艾美耳球虫的发育生物学提供了新见解

New insights into developmental biology of Eimeria tenella revealed by comparative analysis of mRNA N6-methyladenosine modification between unsporulated oocysts and sporulated oocysts

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