Systematic and quantitative analysis of stop codon readthrough in Rett syndrome nonsense mutations

Abstract

Rett syndrome (RTT) is a neurodevelopmental disorder resulting from genetic mutations in the methyl CpG binding protein 2 (MeCP2) gene. Specifically, around 35% of RTT patients harbor premature termination codons (PTCs) within the MeCP2 gene due to nonsense mutations. A promising therapeutic avenue for these individuals involves the use of aminoglycosides, which stimulate translational readthrough (TR) by causing stop codons to be interpreted as sense codons. However, the effectiveness of this treatment depends on several factors, including the type of stop codon and the surrounding nucleotides, collectively referred to as the stop codon context (SCC). Here, we develop a high-content reporter system to precisely measure TR efficiency at different SCCs, assess the recovery of the full-length MeCP2 protein, and evaluate its subcellular localization. We have conducted a comprehensive investigation into the intricate relationship between SCC characteristics and TR induction, examining a total of 14 pathogenic MeCP2 nonsense mutations with the aim to advance the prospects of personalized therapy for individuals with RTT. Our results demonstrate that TR induction can successfully restore full-length MeCP2 protein, albeit to varying degrees, contingent upon the SCC and the specific position of the PTC within the MeCP2 mRNA. TR induction can lead to the re-establishment of nuclear localization of MeCP2, indicating the potential restoration of protein functionality. In summary, our findings underscore the significance of SCC-specific approaches in the development of tailored therapies for RTT. By unraveling the relationship between SCC and TR therapy, we pave the way for personalized, individualized treatment strategies that hold promise for improving the lives of individuals affected by this debilitating neurodevelopmental disorder. KEY MESSAGES: The efficiency of readthrough induction at MeCP2 premature termination codons strongly depends on the stop codon context. The position of the premature termination codon on the transcript influences the readthrough inducibility. A new high-content dual reporter assay facilitates the measurement and prediction of readthrough efficiency of specific nucleotide stop contexts. Readthrough induction results in the recovery of full-length MeCP2 and its re-localization to the nucleus. MeCP2 requires only one of its annotated nuclear localization signals.

Keywords: MeCP2; Aminoglycoside; Personalized medicine; Rare disease; Rett syndrome; Translational readthrough.

Fig. 1

Pathological PTCs in the MeCP2 coding sequence. NTD N-terminal domain, MBD methyl-binding domain, ID intervening domain, TRD transcriptional repression domain, CTD C-terminal domain; frequencies according to RettBASE

Fig. 2

Comparison between the previously established dual reporter system for TR analysis and the novel high-content flow cytometry assay used in this work. a Dual reporter constructs for the analysis of TR. b The high-content assay allows the analysis of samples in a 96-well plate compared to a 12-well plate. The flow cytometer enables direct and automated measurement in the same 96-well cell culture plate which reduces time wasted by transferring each sample and material waste. c Pearson’s correlation and Lin’s concordance correlation coefficient (CCC) as well as d Bland-Altman analysis for TR efficiency measurements between the high-content TR assay and the previously established TR assay

Fig. 3

Basal and induced TR depend on the stop codon type and the context of the PTC. a Representative plot of individual HeLa cells after flow cytometry. Red squares gate untransfected HeLa cells with RFP signals < 700 and GFP signals < 100. Ten thousand events were recorded for each independent measurement. b, c Readthrough was estimated by the ratio of GFP to RFP which was normalized to the same ratio of a 100% TR control. Three replicates each were measured in three independent experiments. b Flow cytometric analysis shows that basal TR of each PTC is generally low (< 1%) except for p.R270X and p.R198X (n = 9). c TR efficiency after induction by geneticin at a concentration of 100 ng/µL depends on the SCC (n = 9, n = 6 for Q244X). Bar plots indicate mean; error bars indicate s.d. One-way ANOVA, with post hoc P values from Bonferroni’s test. *P < 0.033, **P < 0.002, and ***P < 0.001 vs R270X or R198X, respectively

Fig. 4

Recovery of full-length MeCP2 by geneticin treatment. a N-terminal 3XFlag-tagged full-length MeCP2 bearing individual pathological PTCs (red lines). b–d Western blots of MeCP2 mutants using anti-Flag antibody after TR induction sorted by their PTC identity. After transfection, cells were treated for 24 h with 100 ng/µL geneticin. e Quantification of MeCP2 PTC readthrough without or with geneticin treatment, respectively. Readthrough corresponds to the full-length MeCP2 signal intensity normalized to the intensity of full-length plus truncated MeCP2. Bar plots indicate mean; error bars indicate s.d. One-way ANOVA, with post hoc P values from Bonferroni’s test. *P < 0.033, **P < 0.002, and ***P < 0.001 vs. R198X. f Relative level of PTC-containing MeCP2 proteins. The protein level corresponds to the PTC-containing MeCP2 signal intensity normalized to the intensity of wildtype MeCP2 displayed on the same Western blot. Dots indicate mean, error bars indicate s.d. One-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.005 vs. R.168X or R270X, respectively. n = 3 to 4 investigated, independent cell lysates

Fig. 5

Subcellular localization of wildtype and PTC-bearing MeCP2 upon geneticin treatment. a Constructs used for subcellular localization experiments. N-terminally 3XFlag-tagged MeCP2 constructs bear no mutation or one of the following: p.S68X, p.Y141X, p.R198X, p.Q244X, and p.Q406X (red lines). Blue bars indicate the NLS positions. b–e MeCP2 localization of transfected HeLa cells was investigated by immunostaining and fluorescence microscopy. Cells were untreated or treated with 100 ng/µL geneticin for 24 h before fixing. A histogram shows Flag (red) and DAPI (blue) signals along the yellow drawn arrow. b MeCP2 p.S68X and MeCP2 p.Y141X lack both NLSs shifting the nuclear to a cytoplasmic localization. Treatment with geneticin partially recovers the nuclear localization of MeCP2. c MeCP2 p.R198X and MeCP2 p.Q244X bear PTCs between both NLSs and localize to the nucleus without and with geneticin treatment, respectively. d MeCP2 p.Q406X contains a PTC downstream of both NLSs and also localizes to the nucleus without and with geneticin treatment, respectively. Scale bar 10 µm. e Wildtype MeCP2 completely localizes to the nucleus. N > 10 transfected cells in two independent immunofluorescence measurements