Functional Impact of CYFIP2 RNA Editing on Actin Regulation, Axon Growth, and Spinogenesis

Abstract

Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) a component of the Wave Regulatory Complex (WRC), one of the most important players in regulating cellular actin dynamics. Interestingly, CYFIP2 transcript undergoes RNA editing, an epitranscriptomic modification catalysed by ADAR enzymes, that leads 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 aim at investigating the potential implication of CYFIP2 RNA editing related to actin dynamics during cell differentiation, axon development and synaptogenesis in neural cells. We have generated SH-SY5Y neuroblastoma cell lines in which CYFIP2 gene has been functionally inactivated by CRISPR-Cas9 technology. CYFIP2 KO cells showed profound actin filaments disorganisation and loss of the capability to differentiate into a neuronal-like phenotype. Overexpression of both CYFIP2 unedited (K) and edited (E) isoforms rescued normal capability. Finally, we took advantage of primary neuronal culture where endogenous CYFIP2 was knocked down by shRNA technology and CYFIP2 editing variants were overexpressed. While CYFIP2 KD cells reported a decrease in axon development and spine frequency, CYFIP2-E variants increase the number of axon branches, total axon length and dendritic spine frequency compared to either CYFIP2 KD cells or CYFIP-K variants. Overall, our work reveals for the first time a functional significance of the CYFIP2 K/E RNA editing process in regulating the spreading of neuronal axons during the initial stages of in-vitro development and the process of spinogenesis.