Genomic and Transcriptomic Analysis of Ninein Alternative Splicing Between C57BL/6J and DBA/2J Mice

Ethanols anxiolytic actions contribute to increased consumption and the development of Alcohol Use Disorder (AUD). Our laboratory previously identified genetic loci contributing to the anxiolytic-like properties of ethanol in BXD recombinant inbred mice, derived from C57BL/6J (B6) and DBA/2J (D2) progenitor strains. That work identified Ninein (Nin) as a candidate gene underlying ethanols acute anxiolytic-like properties in BXD mice. Nin has a complex exonic content with known alternative splicing events that alter cellular distribution of the NIN protein. We hypothesize that strain-specific differences in Nin alternative splicing contribute to changes in Nin gene expression and B6/D2 strain differences in ethanol anxiolysis. Using quantitative reverse-transcriptase PCR to target Nin alternative splicing, we identified isoform-specific exon expression differences between B6 and D2 mice in prefrontal cortex, nucleus accumbens and amygdala. We extended this analysis using deep RNA sequencing in B6 and D2 nucleus accumbens samples and that Nin expression was significantly higher in D2 mice. Furthermore, exon utilization and alternative splicing analyses identified 8 differentially utilized exons and significant exon-skipping events between the strains, including 3 novel splicing events in the 3 end of the Nin gene that were specific to the D2 strain. Our studies provide the first in-depth analysis of Nin alternative splicing in brain and identify a potential genetic mechanism altering Nin expression between B6 and D2 mice, thus contributing to differences in the anxiolytic-like properties of ethanol between these strains. This work contributes to our understanding of genetic differences modulating ethanol actions on anxiety that may contribute to the risk for alcohol use disorder. Ethanol Naive male C57BL/6J and DBA/2J mice were used to explore basal strain differences in Ninein Alternative Splicing. NAc tissue was harvested and gene expression assessed by Illumina NextSeq2000 RNAseq.乙醇的抗焦虑作用可促进饮酒量增加并推动酒精使用障碍（Alcohol Use Disorder, AUD）的发生发展。本实验室此前在源自C57BL/6J（B6）与DBA/2J（D2）亲本品系的BXD重组近交系小鼠中，鉴定出了与乙醇抗焦虑样特性相关的遗传位点。该研究将九蛋白（Ninein, Nin）鉴定为BXD小鼠中乙醇急性抗焦虑样特性的候选基因。Nin基因具有复杂的外显子组成，且存在已被报道的可变剪接事件，这些事件可改变NIN蛋白的细胞分布。我们提出假说：Nin可变剪接的品系特异性差异，会导致Nin基因表达的变化，以及B6与D2品系小鼠在乙醇抗焦虑效应上的差异。我们通过靶向Nin可变剪接的定量逆转录聚合酶链反应（quantitative reverse-transcriptase PCR），在B6与D2小鼠的前额叶皮层、伏隔核（nucleus accumbens, NAc）及杏仁核中，鉴定出了品系间的亚型特异性外显子表达差异。我们进一步利用深度RNA测序对B6与D2小鼠的伏隔核样本开展分析，发现D2小鼠的Nin基因表达水平显著更高。此外，外显子利用与可变剪接分析共鉴定出8个存在差异利用的外显子，以及品系间显著的外显子跳跃事件，其中包括3个仅在D2品系中出现的Nin基因3'端新型剪接事件。本研究首次对大脑组织中的Nin可变剪接开展了深入分析，鉴定出了B6与D2小鼠间调控Nin基因表达的潜在遗传机制，从而解释了二者在乙醇抗焦虑样特性上的差异。该研究有助于我们理解调控乙醇抗焦虑效应的遗传差异，这类差异可能会影响酒精使用障碍的发病风险。本研究使用了乙醇未暴露的雄性C57BL/6J与DBA/2J小鼠，以探究九蛋白可变剪接的基础品系差异。研究采集了伏隔核组织，并通过因美纳（Illumina）NextSeq2000平台完成RNA测序以评估基因表达。