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 conditionally deleting n-Tr20 in either inhibitory or
excitatory neurons during or post-development. We will also investigate the effect of genetically increasing
mTOR signaling in n-Tr20-/- neurons on synaptic transmission. To further understand these physiological
changes, we will analyze the translatome in excitatory and inhibitory neurons of n-Tr20-/- and wild-type mice and
determine whether n-Tr20 deletion disrupts local translation. In Aim 2, we will test our hypothesis that phenotypes
derived from tRNA loss are due to the decreased level of the pool of tRNAs with the same anticodon, and we
will investigate whether the identity of the depleted tRNA family impacts these phenotypes. We will perform ChIP-
Seq from several major cell types in the brain, utilizing a novel mouse model that can conditionally express an
epitope-tagged allele of RNA Polymerase III. Based on this data, we will identify and delete other highly
expressed tRNAs and investigate the effect of their loss on major cell types in the mouse brain. Finally, we will
extend our work into humans by investigating the impact of tRNA loss on the translatome and physiology of
iPSC-derived neurons.
