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. Overall design: 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.

转运RNA（transfer RNA, tRNA）生物发生缺陷与多种神经系统疾病密切相关，但由于无法在复杂组织的单个细胞类型中精准测定tRNA表达水平，学界对这类疾病的认知始终受到限制。本研究构建了一种Cre依赖型条件性表位标记RNA聚合酶III（RNA Polymerase III）的小鼠模型，可实现在体内对任意细胞类型的tRNA表达进行精准表达谱分析。我们对11种不同的神经系统细胞类型的tRNA表达开展研究，揭示了不同细胞群之间tRNA基因表达的显著异质性。研究发现，尽管维持tRNA反密码子库的水平对细胞稳态至关重要，但神经元对不同tRNA反密码子家族的损伤易感性存在显著差异。细胞类型特异性翻译组（translatome）分析表明，tRNA可用性与密码子需求之间的平衡或许是这种差异化抗损伤能力的潜在机制。本研究为探究大脑中信使RNA（messenger RNA, mRNA）翻译过程与tRNA生物学的复杂机制提供了研究平台。

整体实验设计：将Polr3a-cKI小鼠品系与合适的Cre驱动工具小鼠杂交，获得在对应细胞群体中表达FLAG标记POLR3A的Polr3a Fl/+、CRE+小鼠。本研究共分析了11种中枢神经系统（central nervous system, CNS）细胞类型。此外，我们还对全局敲入小鼠（Polr3A FLAG/FLAG）的全肝（n=2）和小脑（n=4）进行了染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验，该类小鼠在所有细胞类型中均表达FLAG标记的POLR3A。使用FLAG抗体（Sigma, F1804）与Protein G Dynabeads磁珠，从6周龄小鼠的对应脑区中免疫沉淀FLAG标记的POLR3A及其结合的DNA。按照制造商的实验方案，使用NEBNext Ultra II文库制备试剂盒（NEB E7645S）与Illumina平台专用KAPA单端索引接头（Roche, KR1317）构建DNA文库。本实验设计包含免疫沉淀的两个生物学重复，以及每个细胞类型的一个输入对照文库。