Compartment and tissue-specific functions of RNA uridylation

RNA is a remarkably versatile macromolecule that is essential to all life and enables novel therapeutic approaches, such as mRNA vaccines. Cellular RNA molecules come in different flavors and fulfill distinct roles within the cell. Messenger RNA (mRNA), for example, carries genetic information from the site of storage our DNA to the locations of protein synthesis, while many classes of non-coding RNAs (ncRNAs) fulfill a broad range of structural, catalytic and regulatory roles. To function correctly, virtually all RNA molecules are subjected to chemical modification that critically regulate their structural characteristic and functional properties. These RNA modifications are of crucial importance in biological processes like the development of an organism, but also impact disease progression and medical therapy. One noteworthy modification is RNA uridylation, which is carried out by enzymes called terminal uridylyltransferases (TUTases) that specifically target a subset of RNA species to modify their functions and properties. RNA uridylation can, for example, act in cellular quality control and trigger the destruction of faulty ncRNAs. How these molecular functions of uridylation are linked to processes on the organism level remains largely obscure. Importantly, TUTase mutants are linked to human disease and exhibit defects in animal models, including impaired fertility, but it remains unknown why. The fruit fly Drosophila is an ideal system for dissecting the functional consequences of uridylation and TUTase activity due to its genetic resources (including TUTase mutant lines), short generation time, and well-characterized biology. By employing this model system, I aim to elucidate the mechanisms underlying RNA uridylation, investigate its impact on RNA regulation, and understand the in vivo functions using TUTase mutants. My research focuses on a newly identified putative TUTase named Mkg-p. Studying Mkg-p in vivo, alongside other TUTases, will shed light on the molecular mechanisms of RNA uridylation and its importance in cellular processes. To this end, I will use biochemical and high-throughput sequencing approaches to reveal the enzymatic activities of Mkg-p and identify the RNAs that are uridylated by Mkg-p. By using cutting-edge mass spectrometry techniques in flies, I will also discover the molecular interaction partners of Mkg-p. Additionally, I seek to examine uridylation in fruit fly testes, where high levels of TUTases suggest an important role of uridylation in fertility. By investigating the relationship between Mkg-p and other TUTases across different cellular contexts, this research project will provide valuable insights into the intricate world of RNA uridylation and its impact on vital biological processes, including fertility.