Global analysis of excitotoxicity-induced alterations in RNA structure and RNA-protein binding in neurons identifies subsets of transcripts and RNA-binding proteins involved in synaptic plasticity and neurodegenerative disorders [RNA-seq]

Excitotoxicity and altered RNA regulation by RNA-binding proteins (RBPs) are two prevalent hallmarks in multiple neurodegenerative disorders. However, global analysis of RNA-protein interactions in primary neurons in the context of excitotoxicity has not been assessed. To address this, in this work we have applied the protein interaction profile sequencing (PIP-seq) method to primary neurons treated with NMDA to induce excitotoxicity. Our results reveal that NMDA dramatically changes RNA secondary structure at the UTR regions, and that specific RNA structure reduction at the 3’UTR correlates with increased mRNA abundance as assessed by mRNA-Seq. Protein binding sites increase in response to excitotoxicity and, interestingly, different RNA-protein binding in this context defines subsets of transcripts functionally associated with synaptic functions and neurodegenerative disorders. We also identify two RNA motifs enriched in protein binding in the context of excitotoxicity and a list of proteins binding to them in vitro. Accordingly, we observe that two of these RBPs, CELF6 and YBX3 alter their protein levels in response to NMDA treatment, suggesting their involvement in RNA regulation in the context of excitotoxicity. Overall, we describe for the first time the global RNA-protein interactions of primary cortical neurons in baseline conditions and in an in vitro model of excitotoxicity, providing extensive datasets that can be leveraged to bridge the mechanistic gap between two major hallmarks common in multiple neurodegenerative disorders: excitotoxicity and RNA regulation by RBPs兴奋性毒性（Excitotoxicity）与RNA结合蛋白（RNA-binding proteins, RBPs）介导的RNA调控异常，是多种神经退行性疾病中普遍存在的两大标志性病理特征。然而，目前尚未有研究在兴奋性毒性背景下对原代神经元内的RNA-蛋白质相互作用开展全局系统性分析。为填补这一研究空白，本研究采用蛋白质相互作用谱测序（protein interaction profile sequencing, PIP-seq）技术，对经N-甲基-D-天冬氨酸（NMDA）处理以诱导兴奋性毒性的原代神经元进行了检测分析。研究结果显示，NMDA可显著改变非翻译区（Untranslated Region, UTR）的RNA二级结构；且3'非翻译区的特定RNA结构丰度降低，与mRNA测序（mRNA-Seq）检测到的mRNA表达量升高呈显著相关。兴奋性毒性刺激下，蛋白质结合位点的数量显著增加；值得关注的是，此背景下差异化的RNA-蛋白质结合模式，可界定出功能上与突触功能及神经退行性疾病密切相关的转录本子集。本研究还鉴定出两类在兴奋性毒性背景下富集于蛋白质结合区域的RNA基序（RNA motifs），并筛选出一批在体外可与之特异性结合的蛋白质。进一步实验观察发现，其中两种RBPs——CELF6与YBX3，其蛋白表达水平会随NMDA处理发生明显改变，提示二者参与了兴奋性毒性背景下的RNA调控过程。综上，本研究首次全面解析了基础状态下以及兴奋性毒性体外模型中原代皮层神经元的全局RNA-蛋白质相互作用图谱，并提供了丰富的研究数据集，可为弥合多种神经退行性疾病共有的两大核心病理特征——兴奋性毒性与RBPs介导的RNA调控——之间的机制鸿沟提供关键支撑。