PROJECT SUMMARY The post-transcriptional modification of RNA nucleosides is essential for protein production, and therefore life, in all organisms. The chemical modifications to the RNAs that make up the protein synthesis machinery - ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA) - offer cells a mechanism to rapidly and directly control of protein production at multiple steps. Over half a century of modification enzyme knockout studies, coupled with recent efforts to map RNA modifications transcriptome wide, reveal that these chemical additions impact every step in the life cycle of an RNA and help explain their conservation throughout biology. As such, it is unsurprising that the mutation or dysregulation of RNA modifying enzymes is associated with a myriad of negative human health outcomes, including intellectual disabilities, mitochondrial diseases and cancers. However, the molecular mechanisms linking modification dysregulation to human diseases are ill- defined. This is in largely due to the lack of functional roles determined for the vast majority of modified RNA sites. No mechanistic consequences have been identified for > 99% of modified mRNA sites, and < 30% of modified positions in tRNAs have an assigned biological function. Our work will establish relationships between cellular components that shape the RNA modification landscape, discover the impacts of discrete tRNA and mRNA modifications on translation, and uncover a role for one of the most abundant modifications, pseudouridine (), in cellular responses to stress. Modified RNAs are also the foundation of mRNA therapeutics, a new and rapidly expanding class of medical intervention. Altogether, these studies will (i) expand our knowledge of a fundamental mechanism of gene regulation, (ii) inform the design of effective therapeutic mRNAs, and (iii) illuminate the molecular etiology of numerous human diseases.