Functional impact of CYFIP2 RNA editing on actin regulation, axon growth, and spinogenesis

Cytoplasmic FMRP-interacting protein 2 (CYFIP2) is a component of the wave regulatory complex (WRC), one of the most important players in regulating cellular actin dynamics. Interestingly, the CYFIP2 transcript undergoes RNA editing, an epitranscriptomic modification catalyzed by ADAR enzymes, which leads to adenosine (A) to inosine (I) deamination. CYFIP2 editing in the coding sequence results in a K/E substitution at amino acid 320. The functional meaning of this regulation is still unknown. In this study, we aimed to investigate the potential implications of CYFIP2 RNA editing related to actin dynamics during cell differentiation, axon development and synaptogenesis in neural cells. We generated SH-SY5Y neuroblastoma cell lines in which the CYFIP2 gene has been functionally inactivated via CRISPR-Cas9 technology. CYFIP2 KO cells exhibited profound actin filament disorganization and loss of the ability to differentiate into a neuron-like phenotype. The overexpression of both the unedited (K) and edited (E) CYFIP2 isoforms restored normal abilities. Finally, we used primary neuronal cultures in which endogenous CYFIP2 was knocked down via short hairpin RNA (shRNA) technology and CYFIP2 editing variants were overexpressed. While CYFIP2-KD cells presented a decrease in axon development and spine frequency, CYFIP2-E variants increased the number of axon branches, total axon length and dendritic spine frequency compared with both CYFIP2-KD cells and CYFIP-K variants. Overall, our work reveals for the first time the functional significance of the CYFIP2 K/E RNA editing process in regulating the spread of neuronal axons during the initial stages of in vitro development and spinogenesis.