School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan 2308, Australia.
Centre for Brain and Mental Health Research, Hunter Medical Research Institute, New Lambton 2305, Australia.
Int J Mol Sci. 2020 Sep 25;21(19):7086. doi: 10.3390/ijms21197086.
Experience-dependent changes to neural circuitry are shaped by spatially-restricted activity-dependent mRNA translation. Although the complexity of mRNA translation in neuronal cells is widely appreciated, translational profiles associated with neuronal excitation remain largely uncharacterized, and the associated regulatory mechanisms are poorly understood. Here, we employed ribosome profiling, mRNA sequencing and small RNA sequencing to profile transcriptome-wide changes in mRNA translation after whole cell depolarization of differentiated neuroblast cultures, and investigate the contribution of sequence-specific regulatory mechanisms. Immediately after depolarization, a functional partition between transcriptional and translational responses was uncovered, in which many mRNAs were subjected to significant changes in abundance or ribosomal occupancy, but not both. After an extended (2 h) post-stimulus rest phase, however, these changes became synchronized, suggesting that there are different layers of post-transcriptional regulation which are temporally separated but become coordinated over time. Globally, changes in mRNA abundance and translation were found to be associated with a number of intrinsic mRNA features, including mRNA length, GC% and secondary structures; however, the effect of these factors differed between both post-depolarization time-points. Furthermore, small RNA sequencing revealed that miRNAs and tRNA-derived small RNA fragments were subjected to peak changes in expression immediately after stimulation, during which these molecules were predominantly associated with fluctuations in mRNA abundance, consistent with known regulatory mechanisms. These data suggest that excitation-associated neuronal translation is subjected to extensive temporal coordination, with substantial contributions from a number of sequence-dependent regulatory mechanisms.
经验依赖性的神经回路变化是由空间限制的活动依赖性 mRNA 翻译塑造的。尽管神经元细胞中 mRNA 翻译的复杂性已被广泛认识，但与神经元兴奋相关的翻译谱仍在很大程度上未被描述，相关的调控机制也知之甚少。在这里，我们采用核糖体分析、mRNA 测序和小 RNA 测序技术，在分化的神经细胞培养物全细胞去极化后，对整个转录组的 mRNA 翻译进行了全面的分析，并研究了与序列特异性调控机制相关的翻译谱。在去极化后，我们发现转录和翻译反应之间存在一个功能上的分离，其中许多 mRNA 在丰度或核糖体占有率上发生了显著变化，但并非两者都有。然而，在刺激后的延长（2 小时）休息阶段后，这些变化变得同步，表明存在不同层次的转录后调控，它们在时间上是分开的，但随着时间的推移而协调。总体而言，mRNA 丰度和翻译的变化与许多内在的 mRNA 特征有关，包括 mRNA 长度、GC%和二级结构；然而，这些因素的影响在两个去极化后时间点之间有所不同。此外，小 RNA 测序显示，miRNA 和 tRNA 衍生的小 RNA 片段在刺激后立即表达发生峰值变化，在此期间，这些分子主要与 mRNA 丰度的波动相关，这与已知的调控机制一致。这些数据表明，与兴奋相关的神经元翻译受到广泛的时间协调，受到许多序列依赖性调控机制的大量贡献。
