Project Summary. This R35 MIRA application concerns investigating the structure, function, and regulation of two mRNA methyltransferase enzymes: RNA (guanine-N7) methyltransferase (RNMT) and METTL3/METTL14. The first RNA modification was identified in the early 1950s with the discovery of pseudouridine, followed some years later by the identification of N6-methyladenosine (m6A) and N7-methylguanosine (m7G) in the 1970s. Since then, over 150 RNA modifications have been discovered, with many more expected to be uncovered as more sensitive and better tools for their identification are developed. As these modifications emerge, researchers will need to uncover the protein machinery that writes and erases these marks, determine their biological functions, and discern how they are regulated. Defects in the RNA modifications may lead to diseases such as cancer, cardiovascular abnormalities, viral pathogenesis, or cognitive impairment. Therefore, understanding the “code” and the enzymatic processes that govern the presence of RNA modifications are key to revealing pathophysiological mechanisms and the development of novel therapeutics. The overall goal of our research program is to understand the molecular mechanisms for how post-translational modifications regulate the readers, writers, and erasers of RNA modifications, how these marks impact gene expression, and how defects in this system contribute to disease. Many readers, writers, and erasers of RNA modifications harbor PTMs, but our understanding of their influence on protein function is very limited. Therefore, we will use a combination of protein semi-synthesis, enzymology, structural biology, mass spectrometry, and cell biology strategies to unravel these novel regulatory mechanisms. In the next five years, we plan to investigate the mechanistic basis for how RNMT and the METTL3/METTL14 complex are regulated by PTMs, with a long-term vision of expanding to other readers, writers, and erasers in this family. RNMT catalyzes the methylation of the 5’cap of RNA, which is essential for transcript stability, splicing, nuclear transport, and cap-dependent translation. RNMT and its interacting partner, RAM, are phosphorylated and ubiquitinated; however, the molecular and structural details for their regulation and impact on biology are not well understood. The METTL3/METTL14 complex methylates mRNA to generate N6-methyladenosine, which influences transcript processing, stability, translation, and localization. The METTL3/METTL14 complex is regulated by arginine methylation and ubiquitination; however, very little is known about how they impact the complex’s biochemical and biological behavior. Therefore, we plan to assess the mechanistic and structural basis for how these modifications regulate their biochemical and cellular functions using protein semi-synthesis. Furthermore, we plan to characterize the ubiquitin E3 ligases that target these proteins for degradation and deubiquitinases that reverse this...