Figure 4
Genetic rescue of RPS19+/p.Q126X iPSCs restores 40S ribosomal subunit biogenesis. (A) Gene correction strategy. The constitutively active AAVS1 “safe harbor” locus is shown on the top line and the targeting construct is shown below. cDNA expression cassettes driving expression of WT RPS19 or GFP cDNAs under the chicken actin promoter (CAGG) were inserted by zinc finger-mediated homologous recombination into intron 1 of AAVS1. HA, homologous arms left (L) and right (R); SA-2A-Puro-PA, puromycin drug resistance cassette. (B) Quantitative RT-PCR analysis of AAVS1-targeted iPSCs using primers specific for transgenic RPS19 cDNA. Expression was detected specifically in RPS19+/p.Q126X iPSCs after heterozygous integration of the RPS19 cDNA into the AAVS1 locus. RPS19 expression is normalized to the cyclophilin expression level. (C) Western blot showing RPS19 protein in whole-cell lysates of WT1, RPS19+/p.Q126X parental iPSCs (designated as “ – ”) and RPS19+/p.Q126X clones with AAVS1-integrated GFP or RPS19 transgenes. The ratio of RPS19:actin determined by densitometry is shown relative to the WT1 sample, which was assigned an arbitrary value of 1.0. (D) Western blot showing RPS19 protein in nuclear extracts of WT and RPS19+/p.Q126X clones with AAVS1-integrated GFP or RPS19 transgenes. The RPS19:fibrillarin ratios determined by densitometry are shown relative to the WT1 sample, which was assigned an arbitrary value of 1.0. (E) Representative polysome profiles in RPS19+/p.Q126X iPSC subclones with GFP or WT RPS19 cDNA expression cassettes integrated into the AAVS1 locus. Arrows show direction of the sucrose gradient from less to more dense. (F) Summary of 3 independent experiments quantifying the 40S:60S ribosome subunit ratio in RPS19+/p.Q126X iPSC subclones with GFP or WT RPS19 cDNA expression cassettes integrated into the AAVS1 locus. Experiments were performed as illustrated in panel E. *P < .05. (G) Northern blot analysis using the ITS1 probe (see Figure 1D) in RPS19+/p.Q126X iPSCs with GFP or RPS19 cDNA introduced into the AAVS1 locus. RPS19 rescue of RPS19+/p.Q126X iPSCs specifically alleviates impaired processing of 21S pre-rRNA. The ratios of 21S:18SE RNAs determined by densitometry scanning are shown.

Genetic rescue of RPS19+/p.Q126X iPSCs restores 40S ribosomal subunit biogenesis. (A) Gene correction strategy. The constitutively active AAVS1 “safe harbor” locus is shown on the top line and the targeting construct is shown below. cDNA expression cassettes driving expression of WT RPS19 or GFP cDNAs under the chicken actin promoter (CAGG) were inserted by zinc finger-mediated homologous recombination into intron 1 of AAVS1. HA, homologous arms left (L) and right (R); SA-2A-Puro-PA, puromycin drug resistance cassette. (B) Quantitative RT-PCR analysis of AAVS1-targeted iPSCs using primers specific for transgenic RPS19 cDNA. Expression was detected specifically in RPS19+/p.Q126X iPSCs after heterozygous integration of the RPS19 cDNA into the AAVS1 locus. RPS19 expression is normalized to the cyclophilin expression level. (C) Western blot showing RPS19 protein in whole-cell lysates of WT1, RPS19+/p.Q126X parental iPSCs (designated as “ – ”) and RPS19+/p.Q126X clones with AAVS1-integrated GFP or RPS19 transgenes. The ratio of RPS19:actin determined by densitometry is shown relative to the WT1 sample, which was assigned an arbitrary value of 1.0. (D) Western blot showing RPS19 protein in nuclear extracts of WT and RPS19+/p.Q126X clones with AAVS1-integrated GFP or RPS19 transgenes. The RPS19:fibrillarin ratios determined by densitometry are shown relative to the WT1 sample, which was assigned an arbitrary value of 1.0. (E) Representative polysome profiles in RPS19+/p.Q126X iPSC subclones with GFP or WT RPS19 cDNA expression cassettes integrated into the AAVS1 locus. Arrows show direction of the sucrose gradient from less to more dense. (F) Summary of 3 independent experiments quantifying the 40S:60S ribosome subunit ratio in RPS19+/p.Q126X iPSC subclones with GFP or WT RPS19 cDNA expression cassettes integrated into the AAVS1 locus. Experiments were performed as illustrated in panel E. *P < .05. (G) Northern blot analysis using the ITS1 probe (see Figure 1D) in RPS19+/p.Q126X iPSCs with GFP or RPS19 cDNA introduced into the AAVS1 locus. RPS19 rescue of RPS19+/p.Q126X iPSCs specifically alleviates impaired processing of 21S pre-rRNA. The ratios of 21S:18SE RNAs determined by densitometry scanning are shown.

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