Investigation of RNA-specific base modification pathways

RNA碱基上的化学修饰不仅在研究充分的非编码RNA如转运RNA（tRNA）和核糖体RNA（rRNA）中频繁，而且在mRNA中也发现。mRNAs上最突出的修饰是m6 A，它存在于不同的位置，可以对基因表达产生影响。此外，在mRNA上也发现了少量的m1A、m5 C或pseudoU。然而，鉴定通常是困难的，因为特异性抗体很少可用。因此，产生针对修饰核苷酸的高度特异性单克隆抗体是我们项目的目标之一。在第一个资助期间，我们已经建立了针对m6 A，m26 A，m1A和m5 C的抗体。基于我们已经获得的经验，我们现在将继续产生针对m3 C、m4 C、m1 G、m2 G、m5 U、m1 pseudoU和m3 U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，NAD最近已被证明在原核生物和真核生物中充当5' mRNA帽。碱基甲基化由特定的甲基转移酶催化。例如，由胃L3/14组成的二聚体在不同的靶序列上产生m6 A。在第一个资助期间，我们预测未表征的甲基转移酶L2，4，6和8也可能对RNA起作用。在初步的研究中，我们确实发现胃L 8与RNA相关。我们现在将更详细地描述这些酶。我们将（i）研究哪些修饰被催化，（ii）哪些RNA被修饰，（iii）这些酶使用哪些RNA基序。在第一个资助期内，我们已经成功地生产了重组胃L酶。这些蛋白质现在将用于体外甲基化测定以及结构研究。

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.