Ribaudo, G. | Search bioRxiv

bioRxiv2025

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

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.

cell biology↗

bioRxiv2018

Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

CDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

cancer biology↗