tRNA activity can drive pathological processes in a codon-dependent manner, but these mechanisms have never been explored in the context of cancer vulnerability. Transfer RNAs (tRNAs) can regulate protein synthesis dynamics by determining translation velocity, shaping mRNA translation profiles. Thus, regulation of tRNA metabolism is critical for cellular homeostasis, and misregulation of tRNA modification pathways is associated with human diseases. For example, the tRNA methyltransferase complex METTL1-WDR4 catalyzes the N7-methylguanosine (m7G) modification of a tRNA subset and is dysregulated in human cancers. METTL1-WDR4-induced m7G tRNA methylation is essential to control tRNA folding and stability, which affects the translation of mRNAs harboring relevant cognate codons. Multiple lines of evidence show that METTL1, the catalytic subunit, functions as an oncogene through the stabilization and concomitant accumulation of tRNAs, showing that reprogramming of mRNA translation via changes in the tRNA pool can contribute to oncogenesis. For instance, the neuronal development associated tRNA isoform tRNA-Arg-TCT-4-1 possesses oncogenic characteristics and is highly elevated in multiple types of human cancer. On the other hand, failure to deposit m7G leads to the degradation of tRNAs that have been under-modified, as observed in the Rapid tRNA Decay pathway (RTD) in yeast. Despite the growing awareness of the importance of tRNA quality control in normal physiology, it is unclear whether an analogous RTD or other quality control pathways that govern tRNA function are altered in mammalian systems or cancer. Hence, the proposal goals are two-fold: 1) what are the mechanisms underlying the removal of m7G hypomodified tRNAs? and 2) Is the METTL1 substrate tRNA-Arg-TCT-4-1 a potential cancer vulnerability? First, we aim to unveil the pathways and mechanisms that govern the quality control of tRNAs in cancer. To achieve this, we will measure changes in m7G-modified tRNA levels using Hybridization Chain Reaction RNA FISH and monitor translation efficiencies of fluorescent reporters enriched with m7G tRNA decoded codons after whole genome perturbations. Since lack of m7G modification leads to tRNA degradation, we hypothesize that knocking out quality control factors may rescue m7G tRNA levels. Secondly, we aim to test RNA-Arg-TCT- 4-1 inhibition as a potential cancer therapeutic approach using sarcomas as a model. We will develop strategies to target and inhibit this novel oncogenic mediator, starting with antisense oligonucleotides. Mechanistically, we seek to understand how a single tRNA isoform can support malignant phenotypes and further understand how protein translation is dysregulated in sarcoma. Successful completion of the proposed work is expected to provide insight into the fundamental basis for understanding the role of m7G tRNA modification and its regulation in normal and malignant growth.