N6-Methyladenosine modification of mRNA regulates the transition from 2D to 3D growth in the moss Physcomitrium patens [RNA-seq]

In this study, we identified the main genes of the m6A methyltransferase complex (MTC) in P. patens and we demonstrate that their inactivation by genome editing leads to loss of m6A presence in mRNA and results in defects in the moss life cycle. We detected a delay in the formation of gametophore buds, affecting the transition from 2D to 3D growth and subsequent gametophore formation in all MTC mutant plants. We also observed a defect in spore development, with mutant lines producing reduced-size spores that completely failed to germinate. We performed a genome-wide analysis that revealed several transcripts being affected in the Ppmta background. In addition, we demonstrate that the PpAPB1-4 transcripts, known to be central factors regulating the 2D to 3D transition, are m6A-modified, while in the Ppmta mutant the lack of the m6A mark is associated with a corresponding decrease in their transcript accumulation. Overall design: Total RNA was isolated from Ppmta mutant and wild type lines and subjected to high throughput sequencing. Two replicates are included.

本研究中，我们鉴定了小立碗藓（P. patens）中m6A甲基转移酶复合物（m6A methyltransferase complex, MTC）的核心基因，并证实通过基因组编辑使其失活后，会导致mRNA上的m6A修饰完全丢失，进而引发该苔藓的生活周期出现异常。我们检测到所有MTC突变体植株的配子托芽形成出现延迟，影响了其从二维（2D）向三维（3D）生长的转变过程，以及后续配子托的正常形成。此外，我们还观察到突变体株系存在孢子发育缺陷：其产生的孢子体积减小，且完全无法萌发。我们开展了全基因组分析，结果显示在Ppmta突变背景下，多个转录本的表达受到影响。进一步研究证实，已知的调控二维向三维生长转变的核心因子PpAPB1-4转录本存在m6A修饰；而在Ppmta突变体中，m6A修饰的缺失与其转录本积累量的相应下降显著相关。整体实验设计：从Ppmta突变体和野生型株系中分离总RNA，进行高通量测序，实验包含两个生物学重复。