PROJECT SUMMARY/ABSTRACT Transfer RNAs (tRNAs) are critical adaptor molecules that physically link amino acids to codons, decoding mRNA transcripts during translation. The mammalian genome contains hundreds of tRNA genes which are classified into families based on their anticodon. Each family contains multiple tRNA genes, suggesting that these genes may be buffered against the impact of deleterious mutations. Recently, we have demonstrated that a mutation that impairs processing of n-Tr20, a tRNAArgUCU gene, or its complete loss, alters gene expression and physiological responses at both the cellular and organismal level, despite the existence of four additional, functional tRNAArgUCU genes in the mouse genome. More specifically, loss of this highly expressed, neuron- specific member of the tRNAArgUCU family decreases the susceptibility of mice to seizures and alters the excitatory-inhibitory balance in the hippocampus. Loss of n-Tr20 leads to ribosome stalling on cognate AGA codons, along with changes in the transcriptional and translational landscape, characterized by decreased mTORC1 signaling and activation of the integrated stress response. Transgenic overexpression of the other members of the tRNAArgUCU family genes restored seizure susceptibility, in a manner which correlated with the level of tRNA expression from the transgene, suggesting that the phenotypes in n-Tr20-/- mice are due to a decrease in the tRNAArgUCU neuronal pool, to which n-Tr20 is the major contributor. Our results provide the first demonstration that mutation of an individual member of a multicopy, nuclear-encoded tRNA family can alter the molecular landscape and physiology of neurons and provide an impetus for future investigations of tRNA mutations in the maintenance of cellular homeostasis and in disease. This proposal expands upon our findings in several ways. In Aim 1, we will determine the cellular mechanisms underlying the altered excitatory-inhibitory balance upon n-Tr20 loss by co