Biophysical characterization of Arc protein-RNA interactions.

OBJECTIVES: The activity-regulated cytoskeleton-associated protein (Arc) is essential for synaptic plasticity and memory formation. Arc binds RNA and self-assembles into capsid-like structures reminiscent of retroviral Gag proteins, but the molecular basis of this interaction remains poorly defined. This study sought to characterize the biophysical interactions between Arc protein and its mRNA, focusing on the specificity of mRNA binding and its role in regulating Arc self-assembly. METHODS: Here, we combined biophysical approaches to characterize Arc's RNA binding, assembly behavior, and particle morphology. Transmission electron microscopy was employed to visualize complexes of recombinant human Arc and synthetic Arc mRNA at different mRNA molar ratios. Dynamic light scattering (DLS) quantified RNA-dependent changes in the hydrodynamic size of Arc assemblies, while biolayer interferometry (BLI) determined the binding affinity of Arc for cognate and non-cognate mRNA transcripts. RESULTS: Using recombinant human Arc and synthetic Arc mRNA, we found that RNA binding markedly enlarges Arc complexes. At an optimal mRNA molar ratio of ~1 : 45, Arc forms ~50-nm capsid-like particles, compared with ~30-nm assemblies formed by Arc alone, whereas higher RNA levels promote oversized aggregates. DLS confirmed RNA-dependent particle size growth, and BLI showed that Arc binds its cognate mRNA ~1.5-fold more tightly than a non-cognate transcript. CONCLUSION: The results identify RNA as a crucial cofactor for Arc capsid assembly under physiologically relevant conditions and reveal measurable specificity of Arc for its own mRNA. These findings elucidate how Arc packages its own mRNA, providing insight into Arc-mediated neuronal RNA transfer and advancing the development of Arc-based therapeutic delivery systems.