Levels and functions of non-canonical nucleosides in messenger RNA

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The discovery of reversible modification chemistry acting on nucleotides was a breakthrough discovery and led in the course to a gold rush of publications in epigenetic research. In particular, RNA moved back into focus, when it was discovered that messenger RNA may be more chemically diverse than originally believed. Most prominent is the adenosine modification m6A in this respect. Once introduced into mRNA, it serves an ever growing number of functions by interacting with specific reader proteins. We recently published a proteomic study that reveled completely novel connections, influencing disease relevant pathways not only by attraction of reader proteins, but also by repelling them. In the course of the rush, many other nucleotide modifications were identified in mRNA and also DNA. For example, m1A was found in mRNA and the base m6A in the DNA of a number of higher organisms, like algae, insects, worms and even mammals. Some initial discoveries however, had to be refined. As such it was found that both m1A and m5C are far less abundant in mRNA than originally postulated. The demethylating dioxygenase FTO, which was initially described as targeting m6A was recently found to accept predominantly m6Am like it is found in the cap structure. In addition, we could demonstrate with highly sensitive mass spectrometry that m6dA may not be a native component of mammalian DNA as frequently postulated. We will continue our efforts to verify the occurrence of modified bases in mRNA. Detailed data from our group and recent publications revealed that m62A and ms2i6A are found exclusively in rRNA and mitochondrial RNA, respectively. Nevertheless, the presence of in part highly modified nucleosides in mRNA remains a prime target of research. Therefore we are planning to perform a deep survey of nucleoside modifications in mRNA. We propose to study particularly pseudouridine and the 5’ cap structure in more detail. We will reveal their protein-interactome and unravel the functional mechanisms mediated by these interactions. The results will help us to understand the interplay of chemical modification on mRNA and essential cellular processes like translation into proteins.