Circular RNA circRERE2 knockdown in rat primary cortical neurons [polyA RNA-seq]

Circular RNAs (circRNAs), a diverse class of ncRNAs highly enriched in developing neurons, play roles in local protein synthesis and synaptic plasticity. However, distinguishing functional from non-functional circRNAs is challenged by their abundance, tissue specificity and splicing variability. To address this, we conducted a RNAi knockdown screen targeting 32 highly expressed, conserved circRNAs enriched in dendritic processes. circRERE isoforms emerged as regulators of dendritic synapse density and electrophysiological characteristics. mRNA-seq supports the dysregulation of synaptic genes, particularly miR-128-3p-sensitive transcripts. MiR-128-3p activity and expression are reduced, with circRERE possessing multiple miR-128-3p binding sites, suggesting a protective interaction supported by a rescue of the synaptic phenotype upon miR-128-3p overexpression. Conversely, circRERE overexpression with intact miR-128-3p sites rescued the synaptic phenotype and independently increased miR-128-3p levels. These findings demonstrate the necessity for the broad characterization of circRNAs in the nervous system to comprehensively understand their influence on essential non-coding RNA regulatory networks.环状RNA（circRNAs）是一类多样的非编码RNA（non-coding RNAs, ncRNAs），在发育神经元中高度富集，参与局部蛋白质合成与突触可塑性调控。然而，由于环状RNA丰度高、组织特异性强且剪接变异多样，区分具有功能与无功能的环状RNA仍是一项挑战。为解决这一难题，我们针对32种在树突突起中富集的高表达、保守型环状RNA开展了RNA干扰（RNAi）敲低筛选实验。circRERE剪接异构体被证实为树突突触密度及神经元电生理特性的调控因子。mRNA测序（mRNA-seq）结果显示突触相关基因存在表达失调现象，尤其是受miR-128-3p（microRNA-128-3p）调控的转录本。miR-128-3p的活性与表达水平均出现下调，而circRERE含有多个miR-128-3p结合位点，提示二者存在保护性互作；当miR-128-3p过表达时，突触表型得到挽救，这一结果进一步佐证了该互作机制。反之，当保留完整miR-128-3p结合位点的circRERE过表达时，不仅可挽救突触表型，还可独立上调miR-128-3p的表达水平。本研究结果表明，若要全面解析环状RNA对关键非编码RNA调控网络的影响，需对神经系统中的环状RNA开展广泛的功能鉴定与表征工作。