Characterizing the Role of tRNA Expression and Methylation in Proteomics Shifts of the Suicide Brain

Transfer (t)RNAs have received little attention in regards to their role in normative or diseased brain development and function, though they are dynamic mediators of protein synthesis.  The current proposal focuses on characterizing tRNA expression and modifications in the suicide brain and their role in proteomic outcomes that influence disease state. RNA cytosine methylation (m5C), characterized by the addition of a methyl group on the 5' position of a cytosine in RNA, is a novel epigenetic modification only recently characterized in mammalian tissue and most abundant in tRNAs. We recently used viral overexpression and cre-dependent knockdown of NSUN2, a prominent mammalian tRNA methyltransferase, to elucidate the role of NSUN2-mediated tRNA methylation in adult brain function and behavior, and data suggests that dysregulation of cortical NSUN2 induces bi-directional effects on tRNA methylation and depressive-like behavior (Blaze et al., 2021, Nat Comm). Specifically, Nsun2 ablation produced a potent anti-depressant effect which may be mediated by selective changes in Gly tRNA levels leading to significant decreases in Gly-rich proteins involved in synaptic functioning. These data suggest that tRNA expression and methylation may be an unexplored mechanism of translational control of synaptic proteins that are relevant to depression and its treatment. 

While work in our lab thus far has focused on experimental studies in animal models and altering epitranscriptomics via NSUN2-expression manipulation, tRNA methylation and expression has yet to be explored in the healthy or diseased human brain.  Due to the bi-directional effects on depressive-like behavior in our animal model, we now aim to probe tRNA dysregulationin post-mortem brain samples from patients who died by suicide during an episode of major depressive disorder (MDD) (n=49 suicide/49 healthy comparison/27 MDD only). The current proposal aims to use next generation sequencing to measure tRNA methylation and tRNA expression, and an unbiased proteomic approach to identify tRNA-linked codon content of differentially expressed proteins in the suicide brain.  Preliminary data in a partial cohort of the proposed subjects show increases in Gly tRNA methylation in the suicide brain compared to the comparison group, which corresponds to our previous data from animal models of depression. In addition, preliminary data using unbiased tRNA sequencing via YAMATseq shows an increase in Gly tRNA expression at distinct isodecoders in the suicide brain, potentially related to antidepressant treatment status.  We hypothesize that in the full proposed cohort, increases in tRNA methylation will be linked to altered tRNA expression in the suicide brain and further to codon-specific changes in the suicide proteome. Overall, the proposed studies are highly significant because they 1) will be the first to characterize tRNA methylation in the diseased and healthy human brain and 2) elucidate the role of the tRNA regulome on suicidal outcomes and 3) provide novel associations between proteomics and tRNA content in the suicide brain using computational approaches. By identifying these changes in suicide and comparison human PFC tissue, we will make great strides in elucidating a novel epitranscriptomic mark and downstream consequences for psychiatric outcomes, potentially leading to predictors of suicidality or better treatment options.