Abstract
BACKGROUND: Makorin-2 (MKRN2) is an E3 ubiquitin ligase involved in multiple biological processes, yet its role in neurological disorders remains poorly understood. This study aims to elucidate how MKRN2 regulates the RNA-binding protein CSDE1-a molecule linked to autism-related genes-and to explore the functional implications of this interaction in neurodevelopment. METHODS: Using mass-spectrometry screening, we identified CSDE1 as a direct substrate of MKRN2. Ubiquitination sites were validated through mutagenesis of conserved lysine residues. Liquid-liquid phase separation (LLPS) assays were performed in HEK293 and SH-SY5Y cells, and behavioral phenotypes were assessed in Mkrn2-knockout mice. Statistical analyses included appropriate tests for comparing ubiquitination levels, condensate formation, and social behavior outcomes. RESULTS: MKRN2 mediates CSDE1 ubiquitination at four lysine residues (K81, K91, K208, K727). Deletion of MKRN2 or mutation of these sites abolished ubiquitination. MKRN2 and CSDE1 formed co-localized condensates via LLPS, which was disrupted by functional impairment of either protein. Mkrn2-knockout mice exhibited sex-specific social abnormalities-increased sociability in males and social withdrawal in females-recapitulating autism-spectrum disorder (ASD) heterogeneity. We further identified MARK1 and HNRNPUL2, ASD-associated mRNAs, as ubiquitination-dependent targets of CSDE1, linking aberrant condensate dynamics to synaptic plasticity deficits. CONCLUSION: Our study reveals an LLPS-coupled ubiquitination mechanism by which MKRN2 regulates CSDE1, providing a novel molecular pathway underlying neurodevelopmental disorders. These findings offer new insights for understanding and treating neurological diseases such as ASD.