Project Summary/Abstract RNA modifications are concurrently found on nearly all RNA species and can dramatically alter the properties and expression of RNA. Modifications to mRNA can affect gene expression through several mechanisms such as localization, stability, and translation efficiency. Modifications to tRNAs are known to affect their stability and abundance as well as their decoding and elongation dynamics during translation. Dysregulation of many enzyme-mediated RNA modifications have been causally linked to impaired neurological development, intellectual disabilities, and behavioral abnormalities. There has been a great effort by the field to understand the functional impact of individual modifications in isolated RNAs, but the interplay of both mRNA and tRNA modifications has never been explored. The potential for cross-talk between modifications and RNA species echoes the combinatorial control seen in post-translational modifications. We have recently discovered that two distinct RNA-modifying enzymes, tRNA methyltransferase 10A (TRMT10A) and the mRNA demethylase Fat Mass and Obesity protein (FTO), interact and function together to control gene expression. These findings may represent a novel coregulatory mode of gene regulation accomplished by the coordinated RNA-modifying activity of TRMT10A and FTO. In order to better understand the functional role of this enzyme complex, we will (1) define the biochemical and structural parameters of this RNA-modifying complex and (2) elucidate which mechanism(s) underlie how gene expression is modulated by the activity of FTO-TRMT10A. The first goal will take advantage of the array of biophysical tools and expertise available to the Department of Biochemistry and Biophysics to obtain detailed molecular and structural characteristics of wild-type and mutant FTO-TRMT10A complexes. The second goal will be accomplished by employing a spectrum of molecular biology and biochemistry techniques in relevant cell lines to analyze RNA subcellular localization, stability, and modification profile changes. UPenn is an exceptional environment to support my training and this study, fostering a particularly collaborative campus of researchers that cross many intellectual and technical fields. The countless opportunities to learn techniques, use specialized equipment, form collaborations, and share ideas will continue to enrich my research and training. Overall, the tools, training, and expertise are all in place to successfully accomplish the goals set in this proposed study of the novel coregulation of mRNA and tRNA modifications.