Release factor 3 degradation enhances aminoglycoside-mediated translational readthrough at MECP2 nonsense mutations

Premature termination codons (PTCs) in the MECP2 gene are a major cause of Rett syndrome, resulting in truncated proteins and loss of function. Pharmacological induction of translational readthrough (TR) can restore full-length protein, but clinical translation is hindered by the limited efficiency and toxicity of current inducers. Here, we investigated whether targeted degradation of the eukaryotic Release Factor 3a by the small molecules CC-885 or CC-90009 enhances TR at MECP2 PTCs in HeLa cells. Using a dual-fluorescence reporter assay, we found that neither degrader alone significantly increased TR efficiency. However, cotreatment with the TR inducer G418 produced a synergistic effect, resulting in increased TR compared to G418 alone. Although gentamicin, a less toxic TR inducer, led to only weak TR induction on its own, combination with CC-885 resulted in TR levels similar to G418. Combined CC-885 and G418 treatment enabled recovery of full-length MeCP2 protein as well as proper nuclear localization, consistent with functional recovery of the nuclear localization signal. These findings establish eRF3a degradation as a potential strategy to enhance TR and suggest that the combination of aminoglycosides with release factor degraders could provide a less toxic yet effective therapeutic option for PTC-associated disorders.