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Differences in N6-methyladenosine (m6A) methylation among the three major clonal lineages of Toxoplasma gondii tachyzoites
Changning Wei1, Hui Cao1, Chenxu Li1, Hongyu Song1, Qing Liu1, Xingquan Zhu1, 2#, Wenbin Zheng1#

1 Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China

2 Key Laboratory of Veterinary Public Health of Higher Education of Yunnan Province, College of 

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摘要  刚地弓形虫Toxoplasma gondii)呈全球性分布,可感染人和几乎所有的温血动物,引起人兽共患弓形虫病。弓形虫仅有一个种,但存在有200多种基因型,不同基因型虫株具有不同的地理分布和毒力差异。N6-腺苷酸甲基化N6-methyladenosine, m6A)是mRNAs中丰度最高的表观遗传修饰形式,其涉及mRNAs生物学的多种方面,然而目前尚不清楚弓形虫不同基因型之间的mRNA m6A甲基化修饰的差异。因此,本研究采用RNA测序(RNA-seq)和m6A甲基化测序(MeRIP-seq)技术,来探索弓形虫三个主要克隆谱系(I型、II型和III型)之间mRNA m6A甲基化修饰的差异,并鉴定不同基因型之间主要的差异性表达的甲基化基因。结果表明,在弓形虫RHI型)、ME49II型)和VEGIII型)虫株速殖子的521156074974个基因上分别鉴定出765083597264m6A甲基化修饰峰;大多数的m6A甲基化修饰发生在3'UTR,其次是CDS区,且在3'UTR区发生m6A甲基化修饰的基因具有较高的mRNA丰度。通过RH vs. ME49RH vs. VEGME49 vs. VEG组间对比,在676168553个基因上分别鉴定出735192615个差异甲基化峰。进一步结合不同基因型之间的RNA-seq数据分析发现,在RH vs. ME49RH vs. VEGME49 vs. VEG三个比较组中分别鉴定出17241153差异性表达的甲基化基因,且大多数差异性表达的甲基化基因的m6A修饰水平与mRNA丰度之间呈正相关性。对这些差异性表达的甲基化基因进行GO注释分析发现,其主要参与Golgi apparatus、plasma membrane、signal transduction、RNA processing和catalytic step 2 spliceosome等生物学途径;对这些差异性表达的甲基化基因进行KEGG通路富集分析表明,其主要参与endocytosis、systemic lupus erythematosus和mTOR signaling pathway等信号通路。这些发现揭示了m6A甲基化修饰在弓形虫不同基因型虫株之间存在特异性差异,有助于更好地研究m6A甲基化修饰在弓形虫病理生物学中的作用,阐明弓形虫不同基因型虫株之间毒力差异的分子机制。

Abstract  Toxoplasma gondii is an important zoonotic parasite which has over 200 genotypes worldwide. N6-methyladenosine (m6A) methylation is a common epigenetic modification in messenger RNAs (mRNAs), and has been implicated in many aspects of mRNA biology. However, little is known about the difference in m6A methylation among different genotypes of T. gondii. In the present study, we employed methylated RNA immunoprecipitation sequencing (MeRIP-seq) technology to identify key genes exhibiting m6A methylation in the three major clonal lineages (Types I, II and III) of T. gondii tachyzoites. A total of 7650, 8359 and 7264 m6A peaks were identified in 5211, 5607 and 4974 genes in tachyzoites of RH strain (Type I), ME49 strain (Type II) and VEG strain (Type III), respectively. By comparing RH vs. ME49, RH vs. VEG, and ME49 vs. VEG, 735, 192 and 615 differentially methylated peaks (DMPs) were identified in 676, 168 and 553 genes, respectively. A combined MeRIP-seq and RNA-seq analysis revealed 172, 41 and 153 differentially methylated genes (DMGs) at both the m6A methylation and transcriptional level. Gene ontology term enrichment analysis of the DMPs identified differences related to Golgi apparatus, plasma membrane, signal transduction, RNA processing and catalytic step 2 spliceosome. KEGG pathway enrichment analysis showed that the DMGs are mainly involved in endocytosis, systemic lupus erythematosus and mTOR signaling pathway. These findings reveal genotype-specific differences in m6A methylation, which provide new resources for further investigations of the role of m6A in the pathobiology of T. gondii.
Keywords:  Toxoplasma gondii              toxoplasmosis              MeRIP-seq              RNA-seq              N6-methyladenosine  
Online: 24 April 2024  
Fund: This work was supported by the National Key Research and Development Program of China (2021YFC2300800, 2021YFC2300802 and 2021YFC2300804), the NSFC-Yunnan Joint Fund (U2202201), the Research Fund of Shanxi Province for Introduced High-level Leading Talents (RFSXIHLT202101), the Special Research Fund of Shanxi Agricultural University for High-level Talents (2021XG001) and the Veterinary Public Health Innovation Team of Yunnan Province (202105AE160014). We would like to express our special thanks to Professor Hany M. Elsheikha at the Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, University of Nottingham, UK, for critical comments and constructive suggestions on our manuscript.
About author:  #Correspondence Xingquan Zhu, Tel: +86-354-6286886, E-mail: xingquanzhu1@hotmail.com; Wenbin Zheng, Tel: +86-354-6286166, E-mail: wenbinzheng1@126.com

Cite this article: 

Changning Wei, Hui Cao, Chenxu Li, Hongyu Song, Qing Liu, Xingquan Zhu, Wenbin Zheng. 2024. Differences in N6-methyladenosine (m6A) methylation among the three major clonal lineages of Toxoplasma gondii tachyzoites. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.03.072

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