RNA stability controlled by m6A methylation provides X to autosome compensation in mammals [RNA-seq]

In therian mammals, X-chromosomal genes are expressed only from a single active X chromosome, both in males (XY) as well as females (XX). To compensate for this reduction in dosage compared to the evolutionary ancestral state on two autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation (“Ohno's hypothesis”). However, the existence and mechanism of X-to-autosome dosage compensation are still under debate. Here, we show that dosage compensation is achieved via differential N6-methyladenosine (m6A) RNA modification. X-chromosomal transcripts are reduced in m6A modifications and more stable compared to the autosomal counterparts. Acute depletion of m6A using a small molecule inhibitor differentially affects autosomal and X-chromosomal transcripts across sexes, cell types, tissues and species, resulting in perturbed dosage compensation. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation occurs via epitranscriptomic RNA regulation. Overall design: RNAseq experiments were performed on polyA RNA with 4 biological replicates per condition. For female mESC clones, RNAseq experiments were performed on individual clones in one replicate per condition. Please note that the 'HEK293T cells, DMSO, rep1' sample records have been added on Nov 16, 2023.

在兽亚纲哺乳动物中，无论雄性（XY型）还是雌性（XX型），X染色体基因仅能从单条活性X染色体上表达。相较于进化祖先中两条常染色体的基因剂量状态，单拷贝表达导致了剂量水平的下降，因此有研究提出活性X染色体上的基因应存在剂量补偿效应，即奥诺假说（Ohno's hypothesis）。然而，X染色体-常染色体剂量补偿效应的存在性与具体机制仍存在争议。本研究证实，剂量补偿效应是通过差异化的N6-甲基腺嘌呤（m6A）RNA修饰实现的：与常染色体转录本相比，X染色体转录本的m6A修饰水平更低，且稳定性更强。通过小分子抑制剂急性耗竭m6A，会在不同性别、细胞类型、组织及物种中对常染色体与X染色体转录本产生差异化影响，进而导致剂量补偿效应紊乱。本研究提出，X染色体转录本的高稳定性由较低的m6A水平介导，这表明哺乳动物的剂量补偿效应是通过表观转录组层面的RNA调控实现的。实验整体设计：针对聚腺苷酸RNA（polyA RNA）开展RNA测序（RNAseq）实验，每个实验条件设置4次生物学重复。对于雌性小鼠胚胎干细胞（mESC）克隆，每个实验条件下以单个克隆完成一次重复的RNA测序实验。请注意，'HEK293T细胞（HEK293T cells）、二甲基亚砜（DMSO）、重复1'的样本记录已于2023年11月16日补充。