Fig. 7: Reduced ribosome availability more profoundly affects a select subset of transcripts in differentiating neuroepithelia.
From: A programmed decline in ribosome levels governs human early neurodevelopment
a, Schematic of ribo-seq experiment. b, Volcano plot of change in TE of mutant (V190G) relative to control organoids at day 10. Red, log2(fold change) > 0.7 (higher TE in mutant); blue, log2(fold change) < −0.7 (lower TE in mutant); n = 2 independent batches for bulk RNA-seq, n = 2 control organoids and n = 3 mutant organoids for single-organoid ribo-seq; FDR < 0.2. Differential expression analysed by Genewise Negative Binomial GLM with quasi-likelihood tests (two-sided), adjusted for multiple comparisons using the Benjamini–Hochberg FDR method. c, Gene-set enrichment analysis (GO: cellular components) showing enriched terms among transcripts with decreased TE in mutant organoids. All displayed terms showed significant enrichment (FDR < 0.01) in the ‘Down’ direction from CAMERA-PR testing, indicating these cellular components are overrepresented among genes with reduced TE in mutants compared with controls. d, Violin plot of total transcripts and scatter-plot showing the distribution of TE changes of transcripts whose 5′ UTRs contain 5′ TOP-like motifs, shown across FDR thresholds for differential translation. ****P < 0.0001, n = 12,176 total and 105 TOP mRNAs. Unpaired two-sided t-test with Welch’s correction, P = 7.319627 × 10−58. e, Relative change in ribo-seq cpm (red) and RNA-seq cpm (blue) of mutant (V190G) day 10 organoids compared with controls for selected high-confidence transcripts. f, IF image of day 15 control and mutant (V190G) organoids of VIM (green) and mitochondrial matrix HSP60 (red). Note that the mutant exhibits more aggregated mitochondria. Scale bar, 20 μm. Three independent batch were performed. g, TEM micrographs of control and mutant (both V190G and R72W) day 15 organoids. This analysis shows the variant cells exhibit morphologically defective mitochondria, which are often located in autophagic vesicles. Scale bar, 500 nm; zoomed in scale bar, 200 nm. h, OCRs in control, AIRIMV190G and AIRIMR72W day 15 organoids. Control for V190G, n = 5; V190G, n = 6; control for R72W, n = 6; R72W, n = 6 biological replicates. Error bars show s.e.m. i, Model describing how reduced levels of ribosomes in AIRIM variant cerebral organoids results in decreased translation of key mRNAs encoding translation machinery components and neurogenesis factors. Error bars show s.e.m.
