Liquid-liquid phase separation couples MKRN2-mediated ubiquitination of CSDE1 with neurodevelopmental disorders

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.

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.

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.

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.