RNA demethylation induces increased crop biomass and productivity in the field. RNA demethylation induces increased crop biomass and productivity in the field

We performed the transcriptome-wide m6A profiling (MeRIP) with poly(A) RNA samples isolated from shoots and roots of wild type (Nipp), FTO homogeneous transgenic (FTO) and demethylation activity dead mutant variant of FTO transgenic (FTOmut) rice plants. The m6A motif and the distribution of the detected m6A sites along transcripts were consistent with reported results from previous m6A sequencing studies in plants. We then analyzed the differentially methylated m6A sites in poly(A) RNA species including those transcribed from gene loci and from repetitive elements (repeat RNAs). Compared to Nipp plants, both shoots and roots of FTO transgenic plants had showed more hypomethylated m6A peaks (hypo-m6A, 222 in shoots and 3313 in roots) than hypermethylated m6A peaks (hyper-m6A, 63 in shoots and 127 in roots) in mRNAs, while FTOmut rice had showed more hyper-m6A in shoots (349 for hyper vs 31 for hypo) but more hypo-m6A in roots (291 for hyper vs 380 for hypo) than the WT. The hypomethylated m6A peaks in FTO transgenic plants were highly enriched within 3'UTR. Gene Ontology (GO) analysis of these identified hypomethylated m6A-containing coding genes in both FTO transgenic rice shoots and roots revealed an enrichment for functional annotations related to “cellular homeostatic process”, “one-carbon and small molecule metabolic process”, and “gene expression”. We also found the extent of m6A hypomethylation was more pronounced for repeat RNAs than for mRNA species.The quantitative RNA-seq analysis that included ERCC spike-in controls confirmed that FTO plants do indeed accumulate higher overall levels of poly(A) RNA than do WT shoots and roots. Above all, FTO mediated m6A demethylation in plant mRNA and repeats RNA, leading to the activation of transcription. Overall design: Gene expression and m6A peaks were identified from RNA-seq and m6A-seq data, respectively. Both types of data were from the shoots and roots of wild type, FTO homogeneous transgenic and demethylation activity dead mutant variant of FTO homogeneous transgenic rice in two replicates.

我们以从野生型（日本晴，Nipp）、FTO纯合转基因（FTO）及FTO去甲基化活性失活突变体转基因（FTOmut）水稻植株的地上部与根部分离获得的poly(A)尾RNA（polyadenylated RNA）为样本，实施了全转录组范围m⁶A甲基化分析（methylated RNA immunoprecipitation sequencing, MeRIP）。本次检测得到的m⁶A基序与m⁶A位点在转录本上的分布特征，与已发表的植物m⁶A测序研究结果相符。随后我们对poly(A)尾RNA类群中的差异甲基化m⁶A位点开展了分析，此类RNA涵盖基因座转录本与重复元件转录的重复RNA。与野生型日本晴植株相比，FTO转基因水稻的地上部与根部的信使RNA（messenger RNA, mRNA）中，低甲基化m⁶A峰（hypo-m⁶A，地上部222个、根部3313个）的数量均多于高甲基化m⁶A峰（hyper-m⁶A，地上部63个、根部127个）；而FTOmut水稻相较于野生型，地上部的高甲基化m⁶A峰更多（高甲基化349个、低甲基化31个），根部则低甲基化m⁶A峰占优（高甲基化291个、低甲基化380个）。FTO转基因水稻中低甲基化的m⁶A峰显著富集于3'非翻译区（3' untranslated region, 3'UTR）。对FTO转基因水稻地上部与根部中携带低甲基化m⁶A的编码基因进行基因本体（Gene Ontology, GO）富集分析，发现其功能注释显著富集于"细胞稳态过程"、"一碳及小分子代谢过程"以及"基因表达"相关通路。我们还发现，重复RNA的m⁶A低甲基化程度相较于mRNA更为显著。加入外部RNA控制序列（External RNA Controls Consortium, ERCC）spike-in对照的定量RNA测序（RNA sequencing, RNA-seq）分析证实，FTO转基因植株的poly(A)尾RNA整体水平确实高于野生型植株的地上部与根部。综上，FTO介导了植物mRNA与重复RNA的m⁶A去甲基化，进而激活转录过程。整体实验设计：分别通过RNA-seq与m⁶A-seq鉴定基因表达水平与m⁶A峰。两类测序数据均来源于野生型、FTO纯合转基因以及FTO纯合转基因去甲基化活性失活突变体水稻的地上部与根部，且设置两次生物学重复。