Dynamic nature of RNA modifications and their influence in neurological diseases

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RNA and its modifications are dynamically regulated in cellular stress response. Therefore it is not surprising to find mutations in RNA interacting genes as causes for many diseases. My research focuses on the role of RNA modifications in neuronal diseases and ventures into the new field of epitranscriptomics (also referred to as RNA epigenetics). To answer the question how the dynamic nature of RNA modifications is linked to neuronal diseases, the work programme is subdivided into three objectives which interact and complement one another. In objective 1 we will generate mutant and knock-down cell lines of RNA modifying enzymes (RNA writers) with a known connection to neuronal diseases. We will purify the major RNAs like tRNA, rRNA and mRNA and specific RNAs like tRNA fragments and miRNA to assess their modification profile. Using our new technique, nucleic acid isotope labeling coupled with mass spectrometry (NAIL-MS), we will study the stability of these RNAs in dependence of their modification status. With NAIL-MS we will also analyze the dynamics of RNA modifications in the RNA writer mutants and wildtype cells during stress-exposure. These studies will reveal the mechanism of epitranscriptomic stress-response and potential RNA demodification enzymes (RNA erasers). Comparative proteomics of wildtype and mutant cell lines will be used to analyze the molecular consequences of imbalanced RNA modification.In objective 2 we will study the interactome (RNA readers) of small non-coding RNAs like tRNA fragments which have been found to accumulate in these neuronal diseases. We will identify their interactome and compare the interactome of the native, modified RNAs with the interactome of synthetic, non-modified RNAs. This approach will answer the question which downstream processes are affected by the RNA writer mutations which might be causative to pathogenesis. Another aspect of objective 2 covers the fate of tRNA fragments. Are these RNAs substrate to RNA writers and/or RNA eraser? Furthermore we will study the influence of modification on the cellular distribution of RNAs.Objective 3 covers studies arising from the observations made in objective 1 and 2 and will allow a deeper insight into the epitranscriptome. It will reveal the existence of RNA erasers and identify enzyme candidates responsible for the demethylation/demodification process. In a similar fashion we will screen for novel RNA writers and known RNA writers with yet unknown RNA substrates. With our developed tools and knowledge, we will search for yet unkown small non-coding RNAs which accumulate during stress or in the mutant cell lines and determine their involvement in cell homeostasis.The proposed studies will link the mutations of RNA writers found in patients with neuronal diseases to the currently poorly understood pathogenesis.