MECP2 encodes methyl-CpG-binding protein 2, a transcriptional regulator that binds methylated DNA and recruits proteins involved in transcriptional repression and activation. Pathogenic nonsense mutations introduce premature termination codons that can produce truncated, nonfunctional MeCP2 proteins; some also trigger nonsense-mediated mRNA decay, further reducing expression of functional protein. Nonsense mutations account for about 35% of pathogenic MECP2 variants. Most of the premature termination codons examined in the study lie in the terminal exon and escape canonical nonsense-mediated decay, leaving stable messenger RNA that can potentially be translated through the stop codon when readthrough is induced.

Translational readthrough is the recoding of a stop codon by ribosomal incorporation of a near-cognate transfer RNA, allowing peptide elongation and production of a full-length protein. Its efficiency depends strongly on both the type of stop codon and its surrounding nucleotide context. UGA codons are generally the most amenable to pharmacological readthrough, UAG codons show intermediate responsiveness, and UAA codons are least permissive. A cytosine immediately after a stop codon can further increase inducibility. These context-dependent differences mean that a treatment acting on translation termination may not rescue every MECP2 nonsense mutation equally.

The investigators first used a dual-fluorescence reporter containing 14 clinically relevant MECP2 premature termination codons between an upstream red fluorescent protein and a downstream green fluorescent protein. Readthrough was measured after 24 hours as the green-to-red fluorescence ratio, normalized to a control representing complete readthrough. G418 alone produced responses ranging from approximately 3% to 24% at UGA mutations, about 2% to 5% at UAG mutations and less than 1% at UAA mutations. Addition of either CC-885 or CC-90009 increased G418-associated readthrough at most tested codons, with CC-885 producing the stronger and more broadly significant effects at 1 nanomolar. Neither degrader alone significantly induced readthrough at any of the 14 mutations.

CC-885 degraded eRF3a in a concentration-dependent manner. One nanomolar reduced intracellular eRF3a by 50%, 5 nanomolar produced approximately 95% degradation, and 30 nanomolar made the protein undetectable by Western blotting. The compound did not reduce HeLa-cell viability at concentrations up to 5 nanomolar, whereas 30 nanomolar was markedly cytotoxic. G418 was also dose-dependent in its toxicity, and combining it with at least 1 nanomolar CC-885 increased cytotoxicity when G418 concentrations reached 60 nanograms per microliter or more. Under the study’s experimental conditions, 40 nanograms per microliter G418 plus 5 nanomolar CC-885 was the highest tested combination that did not impair viability.

Detailed dose-response experiments with four representative mutations showed that the benefit of combining CC-885 with G418 depended strongly on stop-codon context. At the relatively permissive UGA p.R198X mutation, readthrough reached as much as 40%. For p.R198X, the tolerated combination of 40 nanograms per microliter G418 and 5 nanomolar CC-885 produced approximately 31% readthrough, compared with about 25% for 100 nanograms per microliter G418 alone. The same combination increased readthrough at p.S68X from approximately 4.5% with 100 nanograms per microliter G418 alone to 15%, and at the UAG p.K175X mutation from about 1.1% to 3.5%. The UAA p.K177X mutation remained unresponsive under the tested conditions. A different UAA mutation, p.S49X, responded only when CC-885 concentrations were raised to cytotoxic levels.

Gentamicin, a less toxic aminoglycoside than G418, produced little readthrough by itself even when tested at ten times the concentration used for G418. The combination of gentamicin with 5 nanomolar CC-885 nevertheless increased readthrough at the two UGA variants examined: approximately 20% at p.R198X and 6% at p.S68X. Both values exceeded the corresponding responses to G418 alone. No detectable enhancement occurred at the UAG p.K175X or UAA p.K177X mutations. These observations suggest that the release-factor degrader can strengthen a weak readthrough-inducing stimulus, but only where the stop-codon context is already relatively permissive.

Protein-level experiments with the p.S68X variant provided a separate test of the reporter findings. Because this mutation creates a truncated protein lacking a downstream nuclear-localization signal, restoration of full-length MeCP2 can be assessed by examining both protein length and localization. G418 at 40 nanograms per microliter recovered approximately 1% full-length MeCP2 relative to wild-type controls, while 100 nanograms per microliter recovered about 4%. Adding CC-885 increased full-length recovery to approximately 4% with 40 nanograms per microliter G418 and to a maximum of about 7% with 100 nanograms per microliter G418. Fluorescence microscopy likewise showed stronger nuclear accumulation after combined treatment, reaching more than 40% with 100 nanograms per microliter G418 plus 5 nanomolar CC-885, and cell fractionation supported this result.

Mechanistically, eRF3a is a GTPase that stimulates release factor eRF1 during stop-codon recognition and peptide release. Lowering eRF3a abundance impairs translation termination and increases the opportunity for near-cognate transfer RNAs to be incorporated at a stop codon. The failure of eRF3a degradation alone to induce substantial readthrough indicates that this intervention does not actively promote stop-codon misreading. Instead, it potentiates the aminoglycoside-induced process, which is consistent with a role for reduced termination efficiency only after translation has been perturbed. Readthrough also continued to rise at CC-885 concentrations above 30 nanomolar, even though eRF3a was no longer detectable, suggesting that additional, unresolved effects of the compound may contribute at high doses.

Several limitations constrain what can be inferred from the results. All experiments were performed in transfected HeLa cells over 24 hours, and the viability threshold was specific to that cell line and exposure period; responses to CC-885 can vary substantially among cell types. Long-term depletion of eRF3a could affect global protein synthesis, proteome homeostasis and cell-cycle progression, while aminoglycosides themselves have toxicity constraints, including nephrotoxicity and ototoxicity for gentamicin. Immunofluorescence could not distinguish full-length from truncated p.S68X, and the short truncated protein may enter nuclei by passive diffusion, potentially overstating readthrough. Nuclear localization also does not establish that recovered MeCP2 is functionally equivalent to wild-type protein. Future work would therefore need to test safety, tissue-specific efficacy and recovered-protein activity in neuronal or other disease-relevant models before drawing conclusions about a possible therapeutic strategy for premature-termination-codon disorders.

Subject of Research: Cell-based enhancement of translational readthrough at MECP2 nonsense mutations using eRF3a degradation and aminoglycosides

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

Article References: Lebeda, D., Smoljanow, D., Hofhuis, J., & Thoms, S. (2026). Release factor 3 degradation enhances aminoglycoside-mediated translational readthrough at MECP2 nonsense mutations. Journal of Molecular Medicine, 104(1), Article 123. https://doi.org/10.1007/s00109-026-02728-z

Image Credits: AI Generated

DOI: 10.1007/s00109-026-02728-z

Keywords: MECP2, Rett syndrome, translational readthrough, eRF3a degradation, CC-885, aminoglycosides, premature termination codons, nonsense mutations, translation termination, Release, factor, degradation