N6-methyladenosine (m6A) is the most abundant mRNA modification, and controls mRNA stability. m6A distribution varies considerably between and within species. Yet, it is unclear to what extent this variability is driven by changes in genetic sequences (‘cis’) or cellular environments (‘trans’) and via which mechanisms. Here we dissect the determinants governing RNA methylation via interspecies and intraspecies hybrids in yeast and mammalian systems, coupled with massively parallel reporter assays and m6A-QTL reanalysis. We find that m6A evolution and variability is driven primarily in ‘cis’, via two mechanisms: (1) variations altering m6A consensus motifs, and (2) variation impacting mRNA secondary structure. We establish that mutations impacting RNA structure - even when distant from an m6A consensus motif - causally dictate methylation propensity. Finally, we demonstrate that allele-specific differences in m6A levels lead to allele-specific changes in gene expression. Our findings define the determinants governing m6A evolution and diversity and characterize the consequences thereof on gene expression regulation.

中文翻译：

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通过种间和种内杂交剖析引导 m6A 沉积和进化的序列和结构决定因素

N6-甲基腺苷 (m6A) 是最丰富的 mRNA 修饰，并控制 mRNA 稳定性。 m6A 分布在物种之间和物种内差异很大。然而，目前尚不清楚这种变异在多大程度上是由基因序列（“顺式”）或细胞环境（“反式”）的变化驱动的，以及通过何种机制驱动的。在这里，我们通过酵母和哺乳动物系统中的种间和种内杂交，结合大规模平行报告分析和 m6A-QTL 再分析，剖析了控制 RNA 甲基化的决定因素。我们发现 m6A 进化和变异主要通过“顺式”驱动，通过两种机制：（1）改变 m6A 共有基序的变异，以及（2）影响 mRNA 二级结构的变异。我们确定，影响 RNA 结构的突变（即使远离 m6A 共有基序）也会因果决定甲基化倾向。最后，我们证明 m6A 水平的等位基因特异性差异导致基因表达的等位基因特异性变化。我们的研究结果定义了控制 m6A 进化和多样性的决定因素，并描述了其对基因表达调控的影响。