Investigating how RNA modifications regulate adaptive mRNA translation. - The recent strides of epitranscriptome mapping have unveiled the complex landscape of RNA modifications and their essential role in gene expression regulation. Modifications of transfer RNAs (tRNAs) are critical for proper folding, stability, and decoding function, all essential for enabling precise and efficient translation. Dysregulation in tRNA modification pathways and their associated catalytic enzymes has been implicated in the disruption of mRNA translation processes, contributing to the pathogenesis of numerous human diseases. Emerging evidence demonstrates that tRNA modifications are dynamic and can be regulated in response to cellular stress, indicating that they are likely essential to the orchestration of translational programs that maintain cellular homeostasis, thereby preventing disease development. Our overarching goal is to understand how the regulation of human tRNA modifications influences mRNA translation dynamics and their importance in maintaining normal cellular physiology. This proposal is focused on elucidating the roles of TRMT1L, a newly characterized m22G tRNA methyltransferase, which we have shown is essential for the deposition of other modification important for tRNA function and cellular stress response. We uncovered that TRMT1L interacts with the RIX1 ribosome biogenesis complex, indicating a previously unexplored connection with ribosome biogenesis machinery and tRNA modification regulation. The underlying hypothesis of our research is that the RIX1 complex interacts with TRMT1L to regulate a tRNA modification circuit essential for establishing an mRNA translational program in response to stress. In Aim 1, we will test whether the RIX1 complex is essential for TRMT1L-mediated tRNA modifications using a combination of ChIP, molecular interaction assays and tRNA modification analysis. In Aim 2, using a combination of RNA methyltransferase assays, mass spectrometry analysis, and direct tRNA sequencing on the Oxford Nanopore platform, we will test the role of TRMT1L in regulating a new tRNA D-loop modification circuit. We will also assess whether these downstream modifications change in response to stress and how they affect tRNA function. Finally, using an unbiased Ribo-seq approach to profile actively translated mRNA at high resolution, we will investigate changes in the translational landscape regulated by TRMT1L in cells recovering from the oxidative and endoplasmic reticulum (ER) stresses (Aim 3).
