Cell type-specific expression of tRNAs in the brain regulates cellular homeostasis.

Defects in tRNA biogenesis are associated with diverse neurological disorders, yet our understanding of these diseases has been hampered an inability to determine tRNA expression in individual cell types within a complex tissue. Here, we developed a mouse model in which RNA Polymerase III is conditionally epitope-tagged in a Cre-dependent manner, allowing us to accurately profile tRNA expression in any cell type in vivo. We investigated tRNA expression in eleven diverse nervous system cell types, revealing dramatic heterogeneity in the expression of tRNA genes between populations. We found that while maintenance of levels of tRNA anticodon pools is critical for cellular homeostasis, neurons are differentially vulnerable to insults to distinct tRNA anticodon families. Cell type-specific translatome analysis suggests that the balance between tRNA availability and codon demand may underlie such differential resilience. Our work provides a platform for investigating the complexities of mRNA translation and tRNA biology in the brain. The Polr3a-cKI mouse line was crossed to appropriate Cre driver lines to obtain Polr3a Fl/+, CRE + mice, expressing FLAG-tagged POLR3A in the appropriate cell populations. A total of 11 CNS cell types were analyzed. In addition, ChIP was performed on whole liver (n=2) and cerebellum (n=4) from global knock-in mice (Polr3A FLAG/FLAG) mice, expressing FLAG-tagged POLR3A in every cell-type. FLAG-tagged POLR3A and associated DNAs were immunoprecipitated using FLAG antibody (Sigma, F1804) and protein G dynabeads from the appropriate brain regions of 6-week-old mice. DNA libraries were made using the NEBNext Ultra II Library preparation kit (NEB E7645S) and KAPA Single-Indexed Adapters for Illumina (Roche, KR1317) according to manufacturer’s protocol.The design includes two biological replicates for the immunoprecipitation and one input library for each cell type.