RNA m6A methylome of Caenorhabditis elegans

One of the most abundant RNA modifications is N6-methyladenosine (m6A). RNA from all forms of life, including viruses, contain m6A. This modification has been detected in many types of RNAs, such as mRNA, ribosomal RNA, long non-coding RNAs, small nuclear RNAs and microRNAs. Diverse set of proteins have been characterized to methylate, demethylate and specifically bind to this modification in different organisms. C. elegans is a unique model organism with abundant m6A modification, although its genome does not code for orthologs of the well characterized m6A methyltransferase METTL3/METTL14 complex or the demethylases FTO or ALKBH5. Furthermore, orthologs of the YTH family m6A reader proteins seem to be absent from the worm genome as well. To gain insights into how this modification is installed in this organism, we set out to identify enzymes that contribute to the abundant level of m6A in C. elegans. We designed a targeted RNAi screen by which the expression of 22 candidate putative RNA methyltransferase genes are knocked down. We measured global RNA methylation level by HPLC-MS/MS analysis after two generations of RNAi-mediated knock down. The knock down of two candidate methyltransferases resulted in a decrease in global m6A level in total RNA. The first methyltransferase, F33A8.4, is an ortholog of the human ZCCHC4 gene. The second methyltransferase, C38D4.9, is an ortholog of the human METTL5 gene. In order to determine if ZCCHC4 or METTL5 are involved in the deposition of m6A at the mRNA level, m6A-RIP-seq experiments were performed in mRNA derived from WT (N2), ZCCHC4 KO, METTL5 KO and ZCCHC4/METTL5 dKO C. elegans embryos. m6A-RIP-seq of mRNA derived from C elegans embryos

最丰富的RNA修饰之一为N6-甲基腺嘌呤（N6-methyladenosine，m6A）。涵盖病毒在内的所有生命形式的RNA均含有m6A修饰。该修饰已在多种RNA类型中被检测到，包括信使RNA（messenger RNA，mRNA）、核糖体RNA（ribosomal RNA，rRNA）、长链非编码RNA（long non-coding RNA，lncRNA）、小核RNA（small nuclear RNA，snRNA）以及微小RNA（microRNA，miRNA）。不同物种中已有多种蛋白质被鉴定为可对该修饰进行甲基化、去甲基化并特异性结合。秀丽隐杆线虫（Caenorhabditis elegans，C. elegans）是一类独特的模式生物，其体内m6A修饰丰度极高，但其基因组并不编码经典研究中已充分表征的m6A甲基转移酶METTL3/METTL14复合物，亦不编码去甲基化酶FTO或ALKBH5的同源蛋白。此外，YTH家族m6A阅读蛋白的同源蛋白似乎也不存在于该线虫的基因组中。为了阐明该修饰在该生物中的沉积机制，我们着手鉴定参与秀丽隐杆线虫体内高丰度m6A水平形成的酶类。我们设计了靶向RNA干扰（RNA interference，RNAi）筛选方案，对22个候选推定RNA甲基转移酶基因进行表达敲低。在历经两代RNAi介导的基因敲低后，我们通过高效液相色谱-串联质谱（High Performance Liquid Chromatography-Tandem Mass Spectrometry，HPLC-MS/MS）分析检测了全局RNA甲基化水平。结果显示，两个候选甲基转移酶的敲低可导致总RNA中的全局m6A水平显著下降。第一个甲基转移酶为F33A8.4，是人类ZCCHC4基因的同源蛋白；第二个甲基转移酶为C38D4.9，是人类METTL5基因的同源蛋白。为了确定ZCCHC4或METTL5是否参与mRNA水平的m6A沉积过程，我们对野生型（Wild Type，WT，N2品系）、ZCCHC4敲除（Knock Out，KO）、METTL5敲除（KO）以及ZCCHC4/METTL5双敲除（double Knock Out，dKO）的秀丽隐杆线虫胚胎来源的mRNA开展了m6A RNA免疫沉淀测序（m6A-RIP-seq）实验。针对秀丽隐杆线虫胚胎来源mRNA的m6A-RIP-seq