Biological Function of m5C RNA Methylation in Drosophila

For a long time, the information flow from DNA to protein has been conceived as a succession of mechanisms that copyranscriberanslate genetically encoded sequence faithfully from DNA to RNA to protein. Importantly, the very building blocks of these copied sequences (nucleotides as well as amino acids) are also substrates of molecular machinery that can chemically modify their identity post-synthetically. The existence of these modifications, and ultimately their impact on interpretingranslating the underlying sequences, influences the flow of genetically encoded information through the addition of various epigenetic layers that contribute to the regulation of gene expression. Chemical and covalent modification of RNA and DNA molecules can be detected in most organisms in all kingdoms. Importantly, RNA carries 10 times more chemical modifications than DNA. The majority of these modifications involve methylated groups, which underscores the multifunctional use of a rather simple modification in biologically active molecules. For instance, eukaryotic genomes encode multiple (cytosine-5) RNA methyltransferases (RCMTs), but their biological function has remained ill defined for a long time. Recent studies have provided compelling evidence for the notion that (cytosine-5) RNA methylation is an important contributor to diverse processes such as stress responses, innate immunity and proliferation control. Most of the information about the biological function of (cytosine-5) RNA methylation has only been derived from phenotypic analyses of specific RCMT mutations. Importantly, the molecular machineries that regulate the placing, sensing and interpretation of 5-methylcytosines are unknown and remain to be characterized. The focus of this application is the systematic characterization of (cytosine-5) RNA methylation systems in the fruit fly Drosophila melanogaster. Fruit flies encode homologues for most mammalian RCMTs, and therefore provide an easily accessible entry point into the molecular characterization of RNA methylation systems in mammals. Two Drosophila RCMTs, NSun2 (CG6133) and NSun6 (CG11109), will be genetically manipulated to allow characterizing their tissue-specific and subcellular localization, to define their interactions with both protein and RNA binding partners, and to analyse their mutant phenotypes. In addition, tissue-specific RNA methylation analyses will be performed using recently developed technologies such as miCLiP and RNA-bisulfite sequencing. The characterisation of RCMT interactions and RCMT function both under normal and non-standard laboratory (i.e. stress) conditions will provide important information on the substrate specificities and functional dynamics of these highly conserved RCMTs. Understanding the biology of (cytosine-5) RNA methylation systems in Drosophila will be of great importance for elucidating the molecular mechanisms that are associated with aberrant RNA methylation patterns and their consequences for the organism.

The flow of genetic information from DNA to RNA to protein can be modulated at various levels. One fascinating way of changing this information flow is represented by chemical modifications that are placed onto DNA and/or RNA by a network of enzymatic activities. These proteins can respond to changes in the environment resulting in ill-understood effects on their substrate DNAs or RNAs. The results of this project, which focused on a particular class of proteins that add simple chemical groups to RNA or DNA, contributed to a better understanding of the biological function and impact of these enzymes. By using the fruit fly as a model, the project investigated the molecular function of two proteins (X and Y) that modify small RNAs, while revisiting the debated involvement of X in modifying also DNA. The published results showed that: X is not involved in the chemical modification of DNA; X and Y function is important for controlling specific regions in the fly genome that are called repeat elements; X-mediated impact on these elements was only detectable when flies experienced heat stress, which indicated that environmental changes are sensed by X; Y-mediated impact was independent of heat stress, indicating that Y is contributing to this control independently of environmental changes; When X or Y were removed from the organism, specific small RNAs important for protein synthesis were reduced in number or became fragmented, while genome integrity at specific repeat elements was lost. These combined findings uncovered a link between the molecular activity of two RNA modification enzymes, their substrate RNAs, and genome stability. The results are of great relevance for the emerging field of RNA modification research since they revealed that mutations in particular RNA modification enzymes can cause complex phenotypes, the knowledge that is crucially important when determining the biological function of RNA modifications. Importantly, these findings also opened up promising new research angles since the widespread effects of X and Y on genome integrity suggest that RNAs, which are modified by X and Y are influencing the architecture and packaging of genomic material. Further studies focused on the consequences of decreased small RNA stability in organisms without X and Y. In particular, the potential biological impact of specific RNA fragments that arise during the response to stress conditions was addressed. To facilitate mechanistic studies of these RNA fragments, a biochemical pipeline for their purification was developed, which allows counting their actual copy numbers in single cells, determining their chemical modification status, and revealing their binding partners. These published results are important for future studies on the role of RNA fragments that are produced in cells with impaired RNA modification enzymes (such as X or Y).