Investigation of RNA-specific base modification pathways

对RNA碱基的化学修饰不仅在转移RNAs(TRNAs)和核糖体RNAs(RRNAs)等研究较好的非编码RNAs中频繁发生，而且在mRNAs中也存在。对mRNAs最显著的修饰是m6A，它存在于不同的位置，可以影响基因的表达。此外，在mRNAs上也发现了少量的M1A、M5C或伪U。然而，鉴定往往很困难，因为很少有特定的抗体可用。因此，我们的项目的目标之一是产生针对修饰核苷酸的高度特异性的单抗。在第一个资助期内，我们已经建立了针对M6A、M26A、M1A和M5C的抗体。根据我们已经获得的经验，我们现在将继续产生针对M3C、M4C、M1G、M2G、M5U、M1PEROU和M3U的抗体。此外，我们将建立针对烟酰胺-腺嘌呤-二核苷酸(NAD)的抗体，它最近被证明在原核生物和真核生物中作为5‘mRNA帽。碱基甲基化是由特定的甲基转移酶催化的。例如，由METTL3/14组成的二聚体在不同的靶序列上产生m6A。在第一个资助期，我们预测了未知的甲基转移酶METTL2、4、6和8也可能在RNA上发挥作用。在初步研究中，我们确实发现METTL8与RNA有关。我们现在将更详细地描述这些酶的特征。我们将(I)调查哪些修饰是被催化的，(Ii)哪些RNA被修饰，以及(Iii)这些酶使用哪些RNA基序。在第一个资助期内，我们已经成功地产生了重组的METTL酶。这些蛋白质现在将用于体外甲基化分析以及结构研究。

Chemical modifications on RNA bases are not only frequent in well-studied non-coding RNAs such as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) but also found in mRNAs. The most prominent modification on mRNAs is m6A, which is found at various positions and can have effects on gene expression. In addition, smaller amounts of m1A, m5C or pseudoU have been identified on mRNAs as well. The identification, however, is often difficult since specific antibodies are only rarely available. Therefore, it is one of the goals of our project to generate highly specific monoclonal antibodies against modified nucleotides. During the first funding period, we have established antibodies against m6A, m26A, m1A and m5C. Based on the experience we have gained, we will now continue to generate antibodies against m3C, m4C, m1G, m2G, m5U, m1pseudoU and m3U. Furthermore, we will establish antibodies against nicotinamide-adenine-dinucleotide (NAD), which has recently been shown to serve as 5’ mRNA cap in prokaryotes and eukaryotes. Base-methylations are catalyzed by specific methyltransferases. A dimer composed of METTL3/14, for example, generates m6A on distinct target sequences. During the first funding period, we predicted that the uncharacterized methyltransferases METTL2, 4, 6 and 8 might function on RNAs as well. In preliminary studies we indeed found that METTL8 is associated with RNA. We will now characterize these enzymes in more detail. We will (i) investigate which modification is catalyzed, (ii) which RNAs are modified and (iii) which RNA motifs are used by these enzymes. During the first funding period, we already succeeded to generate recombinant METTL enzymes. These proteins will now be used for in vitro methylation assays as well structural studies.