Effects of alcohol and PARP inhibition on RNA ribosomal engagement in cortical excitatory neurons

The objective of the study is to describe the effects of EtOH on excitatory pre-frontal cortical neuronal ribosomal-engagement (RE) transcripts from total-RNA and provides insights into Poly (ADP-ribose) polymerase (PARP)-mediated regulation of EtOH effects (using the PARP inhibitor ABT-888). Mice whose ribosomes were tagged with EGFP expressed only in CaMKIIα expressing cells were used for the study. PFC samples were homogenized in the appropriate buffer and TRAP-RNA (collected by anti-EGFP antibody incubation) and total-RNA samples were extracted. Following quantification and quality control, the samples were subjected to rRNA depletion, library preparation and 50bp paired-end sequencing (NovaSeq 6000) using the Illumina platform. Data were analyzed using standardized bioinformatic packages and normalized based on treatment status, i.e., Control, EtOH or EtOH+ABT-888, as well as by sample type, i.e., Input or TRAP.

本研究旨在阐明乙醇（EtOH）对来源于总RNA的兴奋性前额叶皮层（prefrontal cortex, PFC）神经元核糖体结合转录本（ribosomal-engagement, RE）的影响，并为解析聚(ADP-核糖)聚合酶（Poly (ADP-ribose) polymerase, PARP）介导的乙醇作用调控机制提供实验依据（采用PARP抑制剂ABT-888进行干预）。本研究使用仅在CaMKIIα阳性细胞中表达增强绿色荧光蛋白（enhanced green fluorescent protein, EGFP）以标记核糖体的小鼠作为实验对象。对PFC样本采用适宜缓冲液进行匀浆处理，通过抗EGFP抗体孵育收集TRAP-RNA，并提取总RNA样本。经定量与质量控制后，对样本进行核糖体RNA（ribosomal RNA, rRNA）去除、文库构建，并采用Illumina平台的NovaSeq 6000系统完成50bp双端测序。数据采用标准化生物信息学工具包进行分析，并根据处理分组（对照组、乙醇组、乙醇+ABT-888组）及样本类型（Input组、TRAP组）进行归一化处理。