The Protease Omi Regulates Neuronal Differentiation Through Cleaving E2F1

Aim: Omi is a protease with multiple functions. The aim of this study was to investigate the role of Omi in regulating neuronal differentiation and to identify the substrate of Omi involved in this process.Methods: GST pulldown and in vitro cleavage assays were used to identify Omi substrate. The protein expression was detected using immunoblot analysis. Cell transfection experiments were performed using cells transfected with plasmids, si RNA or sh RNA. Neurite outgrowth was analyzed using Image J to measure the neurite length using N2 a cells that were transfected with sh RNA or Omi inhibitor UCF101, along with a treatment with 20 μM retinoic acid(RA).Results: In mnd2 mice, a Omi protease-deficient strain, and in Omi knockdown cells, E2F1 was up-regulated. However, in cells overexpressing wide type Omi but not the protease-deficient S276 C Omi, E2F1 was decreased. The interactions of Omi and E2F1 were detected in mouse brain as well as transfected cells. In vitro cleavage assay showed that Omi directly cleaved E2F1. Furthermore, in N2 a cells, knockdown of Omi or administration of Omi specific inhibitor UCF101 inhibited neurite outgrowth, accompanied by an increased expression of E2F1.Conclusion: E2F1 is a substrate of Omi. Omi regulates neurite outgrowth by repressing E2F1 in differentiated neuronal cells.