Cytosine methylation as a new mechanism to regulate long non-coding RNAs

It is well established that RNA and DNA are subject to postsynthetic chemical modifications such as methylation. Whereas in DNA mostly cytosines are modified, RNAs contain a large number of diverse modifications that can target all bases and the ribose moiety. The vast majority of modifications, however, has been detected and studied in abundant non-coding RNAs such as tRNAs or rRNAs. In contrast, knowledge about the nature and - even more so - the functions of posttranscriptional modifications in poly(A)RNA is extremely sparse. The deep-sequencing efforts of the past decade have revealed an impressive complexity of the eukaryotic transcriptome, and new RNA species are discovered on a regular basis. A particularly interesting class of RNAs comprises the long non-coding RNAs (lncRNA). These polyadenylated RNAs have been implicated as regulators of chromatin structure and gene activity. Until very recently, no information was available about whether lncRNAs are modification targets and if so, what effects these modifications might have on their function. We and others have recently demonstrated that poly(A)RNAs contain cytosine methylation (m5C). In particular, we found that the lncRNAs HOTAIR and XIST are subject to cytosine methylation. In both RNAs, m5C was found to be located in the vicinity of functionally important protein binding regions. We also discovered that m5C abolished the interaction of XIST with the chromatin modifying PRC2 complex suggesting that cytosine methylation may have a role in regulating the interaction between lncRNAs and their protein binding partners. With this proposal, we plan to continue and extend our studies of cytosine methylation in lncRNAs with the ultimate goal of understanding the biochemical and biological impact of this modification on the functions of regulatory lncRNAs. Specifically, we will determine the global distribution of cytosine methylation in poly(A)RNA and enriched nuclear poly(A)RNA. We will employ in depth bioinformatics analyses to detect correlations with various structural and functional parameters of the target RNAs, and we will investigate the functional impact of cytosine methylation on RNA-protein interaction. We will also seek to identify the RNA methyltransferase enzymes that are responsible for specific methylations. Finally, we will work on the development of new sequencing based methods to identify potentially new cytosine modifications in RNA. With these studies, we hope to uncover novel mechanisms for the regulation of RNA, in particular lncRNA, function. Since the regulation of mRNAs and non-coding RNAs is a fundamental process that affects all areas of life, the results from this work will provide the groundwork for novel insights into processes ranging from cell cycle progression, development, and differentiation to human diseases and disease therapy.

Genetic information stored in the DNA is read and interpreted by many tightly regulated processes. In the first step, DNA is transcribed into RNA. These molecules fulfill diverse functions in the cell ranging from serving as messengers (mRNA) for the generation of protein, acting as regulatory molecules to functioning as RNA enzymes. To ensure optimal fine-tuning of its activity, the RNA itself is subject to various modifications including the attachment or removal of chemical groups, such as methyl groups. Thereby, the sequence information typically is not altered but the modification adds information regarding the subsequent fate of the RNA molecule, which is why this process is frequently referred to as "epitranscriptomic" modifications. The study of the epitranscriptome is an emerging field of research and although great progress has been made in recent years, there are many open questions. In this project, we have investigated if and where methylated cytosine (m5C) can be found in RNA. To this end, we established new experimental and analytical methods to detect m5C. Employing these tools, we elucidated for the first time the distribution of m5C in mRNA of mouse embryonic stem cells and the brain. We also examined the role of several RNA methyltransferase enzymes in this context. Finally, we developed a new method, called TUC-seq, that relies on metabolic labeling of RNA by modified nucleosides and chemical conversion of the labeled RNAs. In this way, TUC-seq allows for simple and straight-forward sequencing-based distinction between newly transcribed and preexisting RNA. We showed that this method is well suited to study RNA synthesis and decay rates. In summary, these studies provided significant progress in understanding epitranscriptomic processes. The regulation of mRNAs and non-coding RNAs is a fundamental process that affects all areas of life. Hence, the results and tools derived from this work will provide the groundwork for future studies addressing the function of RNA modifications in diverse biological contexts including human diseases and disease therapy.