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. 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.

本研究针对野生型（日本晴，Nipp）、FTO纯合转基因（FTO）以及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聚腺苷酸化RNA（poly(A) RNA），开展了全转录组范围的N6-甲基腺嘌呤（m⁶A）甲基化图谱分析（MeRIP）。本研究检测到的m⁶A基序以及转录本上m⁶A位点的分布特征，与此前植物领域m⁶A测序研究的报道结果一致。随后，我们对聚腺苷酸化RNA（poly(A) RNA）物种中的差异甲基化m⁶A位点进行了分析，这些RNA包括来自基因座转录的mRNA以及重复序列元件转录的重复RNA（repeat RNAs）。与日本晴（Nipp）植株相比，FTO转基因水稻的地上部与根部在mRNA中检测到的低甲基化m⁶A峰（hypo-m⁶A，地上部222个、根部3313个）数量显著多于高甲基化m⁶A峰（hyper-m⁶A，地上部63个、根部127个）；而FTOmut水稻与野生型相比，地上部的高甲基化m⁶A峰更多（高甲基化349个vs低甲基化31个），但根部的低甲基化m⁶A峰更多（高甲基化291个vs低甲基化380个）。FTO转基因水稻中的低甲基化m⁶A峰在3'非翻译区（3'UTR）中显著富集。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（GO）富集分析，结果显示其功能注释显著富集于"细胞稳态过程"、"一碳与小分子代谢过程"以及"基因表达"相关通路。我们还发现，重复RNA（repeat RNAs）的m⁶A低甲基化程度较mRNA物种更为显著。纳入外部RNA控制序列（ERCC）spike-in对照的定量RNA测序（RNA-seq）分析证实，FTO转基因水稻的地上部与根部的聚腺苷酸化RNA（poly(A) RNA）总水平确实高于野生型植株。综上，FTO可介导植物mRNA与重复RNA的m⁶A去甲基化，进而激活转录过程。基因表达量与m⁶A峰分别通过RNA测序（RNA-seq）与m⁶A测序（m⁶A-seq）数据鉴定得到。两类数据均来源于野生型、FTO纯合转基因以及FTO去甲基化活性失活突变体转基因水稻的地上部与根部，且设置了两个生物学重复。