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. Overall design: We performed highly penetrant electroporation of primary cortical neurons. We utilised an empty vector control (pSUPER), a ScramSh control (Pum2MM, a mismatched scrambled shRNA) and a shRNA expressing plasmid knocking down circRERE2 (RERE2). There were four biological replicates gathered (N1-N4) of each of the 3 conditions. The same input RNA was used for both miRNA- and polyA-RNA-seq.