Extended Data Fig. 7: Reduced ribosome biogenesis and processing in post-mitotic cells.
From: Pre-rRNA spatial distribution and functional organization of the nucleolus
a. Active rDNA loci are reduced in post-mitotic cells. Top, schematic of rDNA FISH probes. Bottom, representative images of rDNA (grey), FBL (green) and RPA194 (magenta) in H9, D30 arcuate neuron, HeLa and SH-SY5Y cells, showing active rDNA loci colocalizing with Pol I. b. Top, quantification of active rDNA ratios per cell from (a), n = 12 (H9), 18 (neurons), 15 (HeLa), 27 (SH-SY5Y) cells. Bottom, schematic of rDNA organization in FC–DFC units under altered proliferation rates. c. Majority of pre-rRNA processing factors exhibit reduced expression during differentiation. Volcano plot showing differential gene expression (D30 arcuate neurons vs. H9) of 525 annotated pre-rRNA processing factors. Data was analysed using three biological repeats. The p.adjust values were computed by DESeq2, which used a Wald test by default to calculate original p-values then adjusted for multiple testing using the Benjamini-Hochberg method. d. Heatmap of SSU- and LSU-precursor- associated protein enrichment dynamics in SH-SY5Y cells. SSU-precursor-bound proteins with peak enrichment time at early time point (0- and 30 min) and LSU-precursor-bound proteins with peak enrichment time at late time point (60 min) are marked by black boxes. Data was analysed using four MS repeats performed as in Extended Data Fig. 2a in SH-SY5Y cells. Mean log2 LFQ intensities were first averaged across four MS repeats and then z-score normalized across time points. e. Delayed binding of SSU precursor associated proteins in SH-SY5Y cells. LOESS regression was performed on quantities of pre-rRNA-bound proteins and chase time points. The quantities of proteins were derived from MS data of HeLa (Extended Data Fig. 3c) and SH-SY5Y cells (d). f. Proportion of pre-RNA-bound proteins enriched at early time points (0- and 30 min) and at late time point (60 min) in HeLa (derived from Extended Data Fig. 3c) and SH-SY5Y cells (derived from panel (d)). Number of proteins is labelled on the bar. g. Representative averaged SIM images of nascent pre-rRNAs over chase times (0–60 min) in H9, SH-SY5Y and D30 arcuate neuron cells. Each panel represents averaged signal from n = 52, 50, 51, 50, 51 and 51 DFC units per time point (H9 cells); 49, 38, 50, 57, 52 and 50 DFC units per time point (SH-SY5Y cells); 30, 30, 30, 33, 37 and 32 DFC units per time point (in D30 arcuate neurons). Scale bars, 500 nm.
