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. 2016 Mar 7;5:e13722. doi: 10.7554/eLife.13722

Figure 8. Ki-67 is not required for rRNA biogenesis but controls gene transcription.

(A) Northern-blot analysis of total RNA extracted from HeLa and U2OS cells constitutively expressing shRNA against Ki-67; and HeLa, U2OS, HCT-116 and HCT-116 TP53 (-/-) depleted of Ki-67 by siRNA for 72 hr in two biological replicates (#1 and #2) or with scrambled siRNA control (Scr). Pre-rRNA intermediates were analysed by probing with different primers located in the different spacers of the 47S sequence (5’ETS-green; ITS1-blue; ITS2-purple). (B) Quantification of 47S rRNA precursor in HeLa, HCT-116 and HCT-116 TP53 (-/-) depleted of Ki-67 by siRNA for 72 hr in three biological replicates (n=1–3). (C) U2OS cells (left) or HeLa cells (right) show transcriptome profile differences (fold change >1.5; corrected p-value <0.02) between asynchronous cells constitutively expressing control (CTRL) or Ki-67 shRNA. Heat-maps present the expression levels of differentially expressed genes between biological replicates (1,2,3) and technical replicates (3, 3*). Data is provided in Figure 8—source data 1 and 2.

DOI: http://dx.doi.org/10.7554/eLife.13722.032

Figure 8—source data 1. Ki-67-dependent transcriptome in U2OS cells.
Table showing statistically significant (corrected p value < 0.02, Fold-change >1.5) changes of transcript abundance from Agilent Gene chip analysis of cDNA from control U2OS (pGIPZ-shRNA non silencing control) and U2OS stably silenced (pGIPZ-Ki-67 shRNA) for Ki-67.
DOI: http://dx.doi.org/10.7554/eLife.13722.033
elife-13722-fig8-data1.xls (140.6KB, xls)
DOI: 10.7554/eLife.13722.033
Figure 8—source data 2. Ki-67-dependent transcriptome in HeLa cells.
Table showing statistically significant (corrected p value <0.02, Fold-change >1.5) changes of transcript abundance from Agilent Gene chip analysis of cDNA from control HeLa (pGIPZ-shRNA non silencing control) and HeLa stably silenced (pGIPZ-Ki-67 shRNA) for Ki-67.
DOI: http://dx.doi.org/10.7554/eLife.13722.034
elife-13722-fig8-data2.xls (278.6KB, xls)
DOI: 10.7554/eLife.13722.034

Figure 8.

Figure 8—figure supplement 1. Ki-67 depletion does not hinder rRNA transcription.

Figure 8—figure supplement 1.

Newly synthesised RNA in asynchronous U2OS cells expressing control or Ki-67 shRNA visualised by 5-ethynyl uridine (EU) incorporation and immunofluorescence. Bar, 10 µm.
DOI: http://dx.doi.org/10.7554/eLife.13722.035
Figure 8—figure supplement 2. Human pre-rRNA processing pathway involves two major pathways.

Figure 8—figure supplement 2.

The two pathways (A and B) are characterized by specific cleavage kinetics. In pathway A, cleavages at the site A0 and 1 occur before cleavage at the site 2, whereas cleavage at site 2 occurs before cleavages at sites A0 and 1 in the pathway B. Both pathways will end by the production of the mature 28S, 18S and 5.8S. The 5S rRNA is synthesized by the RNA pol III in the nucleoplasm and will join the other rRNAs during ribosome assembly.
DOI: http://dx.doi.org/10.7554/eLife.13722.036