Profiling of N6-methyladenosine dynamics indicates regulation of oyster development by m6A-RNA epitranscriptomes.

The N6-methyladenosine (m6A) has recently emerged as an important layer of the gene expression regulatory network with critical implications in vertebrate and insect development. However, despite an m6A-RNA pathway is present in the pacific oyster Crassostrea gigas, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. We performed RNA sequencing (RNA-seq) and m6A-RNA immunoprecipitation sequencing (MeRIP-seq) on 10 developmental stages, on two distinct developments, to covering the whole oyster developement. The m6A-RNA methylomes show the conservation of m6A consensus motif RRACH, a m6A peak around the stop codon and highlight specific m6A signatures according to RNA species between mRNAs, lncRNAs and transposable elements. The differentially methylated RNAs constitute developmental clusters that correspond to chronological steps of oyster development (cleavage, gastrulation, tissues differentiation and metamorphosis). They are marked with an overall drop of mRNA and lncRNA methylation at the morula stage followed by a global increase up to pre-metamorphosis. Messenger RNA m6A levels are correlated to transcript content and shifts in methyladenine profiles correspond to expression kinetics. The m6A of TE transcripts is also regulated and peaks during the gastrulation. Altogether our results indicate that m6A epitranscriptomes are an important regulator of oyster development. This first epitranscriptome profiling across the development of a lophotrochozoan model brings new insights into the epigenetic control of developmental processes and their evolution. Overall design: Examination of m6A and gene expression levels in 10 embryo-larval to post-metamorphosis stages in the oysterN6-甲基腺嘌呤（N6-methyladenosine，m6A）作为基因表达调控网络的重要层级，近年来被证实对脊椎动物与昆虫发育具有关键调控作用。尽管太平洋牡蛎（Crassostrea gigas）中存在m6A-RNA通路，但担轮动物（lophotrochozoan）类生物的表观转录组（epitranscriptome）发育意义仍未明确。 本研究针对太平洋牡蛎的完整发育周期，选取两个独立发育进程的10个发育阶段，开展RNA测序（RNA-seq）与m6A RNA免疫沉淀测序（MeRIP-seq）。所得m6A RNA甲基化组结果显示：m6A的保守基序为RRACH，其富集峰集中于终止密码子区域；同时可依据RNA类型（信使RNA、长链非编码RNA与转座因子），区分出特异性的m6A修饰特征。 差异甲基化RNA所形成的发育簇，与牡蛎发育的时序进程一一对应，涵盖卵裂、原肠胚形成、组织分化与变态发育等阶段。该类RNA的甲基化整体呈现如下模式：桑椹胚阶段信使RNA与长链非编码RNA的甲基化水平出现整体下降，随后持续升高直至变态前期。信使RNA的m6A水平与转录本丰度呈正相关，甲基腺嘌呤修饰谱的变化与基因表达动力学高度吻合。转座因子转录本的m6A修饰同样受到精准调控，并在原肠胚形成阶段达到峰值。 综上，本研究结果证实m6A表观转录组是调控牡蛎发育的关键因子之一。本研究作为首个针对担轮动物模型全发育周期的表观转录组图谱分析，为发育过程的表观遗传调控机制及其演化提供了全新视角。 实验设计概述：检测太平洋牡蛎10个从胚胎幼虫至变态后阶段的m6A修饰水平与基因表达量。