Fig. 4: The AIRIMV190G variant causes stage-specific defects in protein synthesis and ribosome levels.
From: A programmed decline in ribosome levels governs human early neurodevelopment
a, Experimental schemes of OP-puro based translation capacity and TMT assays. b, Representative images showing the OP-puro signal in control and mutant (V190G) organoids at days 5, 10 and 15. Mutant organoids exhibited lower OP-puro signals than controls starting at day 10. Scale bar, 50 μm. c, Quantification of OPP signal of individual NE bud at day 5, 10 and 15 mutant (V190G) organoids relative to their corresponding control. Dunn’s multiple comparisons test (two-sided). n = 11, 17 and 11 individual EB or NE buds from six individual organoids imaged from two independent batches. Error bars show s.e.m. Day 5 versus day 10 adjusted P value (Padj) = 8.013453 × 10−5; day 5 versus day 15 Padj = 4.692448 × 10−5, **P < 0.01, ***P < 0.001. d, Volcano plots of the −log10-transformed P value versus the log2-transformed ratio of mutant/control organoids at days 5, 10 and 15 (n = 3,539, 3,544 and 3,541 proteins, respectively n = 3 biologically independent samples). Discovery was determined using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, a false discovery rate (FDR)-controlling method for multiple comparisons (two-sided test). e, Violin plots showing the distribution of r-proteins as in d (n = 76 r-proteins; n = 3 biologically independent samples). Adjusted P values were calculated from Tukey’s multiple comparisons test (two-sided). **P < 0.01, ****P < 0.0001. Error bars show s.e.m. f, Polysome profiling with the corresponding western blots comparing the distribution of RSL24D1 and RPL28 in control and AIRIMR72W iPS cells. Three independent batches were performed. g, Polysome profiling with corresponding western blots comparing the distribution of RSL24D1 and RPL28 in control and AIRIMR72W day 15 organoids. Three independent batches were performed. h, IF showing the subcellular distribution of RSL24D1 in control and AIRIMR72W day 15 organoids. Scale bar, 20 μm. Three independent batches were performed. i, Quantification of the nuclear/cytoplasm ratio of RSL24D1 immunofluorescence of control, AIRIMV190G and AIRIMR72W variant day 15 cerebral organoids. *P = 0.032, **P = 0.0042. Dunnett’s T3 multiple comparisons test (two-sided). Control for V190G, n = 8; control for V190G, n = 9; control for R72W, n = 10; control for R72W, n = 13 imaged regions from two independent batches. Error bars show s.e.m.
