Figure 7.
Figure 7. Nle inactivation induces defects in rRNA maturation and accumulation of the large preribosomal subunit. (A) Schematic representation of the two major pre-rRNA processing pathways in mammalian cells. Localization of probes is indicated under the 47S primary transcript. Predictions based on the known function of NLE orthologue in yeast of rRNA maturation defects in Nle-deficient mouse ES cells are shown in red. If this function is conserved, Nle inactivation should induce a block in the last steps of maturation of the large preribosomal subunit, leading to an accumulation in the nucleolus of the 32S and 12S pre-rRNA species and the decreased export in the cytoplasm of mature 28S and 5.8S rRNA. 32S and 12S pre-rRNA can be detected by Northern blot or FISH analysis using probes directed against the pre-rRNA internal sequence its2 (blue). Maturation of the small subunit should not be directly affected. Thereby, the levels of the corresponding pre-rRNA detected with an its1 probe (green) should remain unchanged. Similarly, levels of 47S primary rRNA transcript detected with a 5′ets probe (purple) should not be directly affected. (B–D) Northern blot analysis of total RNA from untreated or treated ES cells. (B) Quantification of 12S/5.8S, 32S/28S, and 30S/18S intensity ratios. Northern blots analysis with 28S, 18S, 5.8S, or its2 probe was obtained at least twice. (E–G) FISH experiment on untreated and treated ES cells. (E) Representative images of two independent FISH experiments with its2, its1, or 5′ets performed on ES cells untreated or treated with 4-OHT at day 3. Cells were labeled with Hoechst. Bar, 5 µm. (F) Representative FACS profiles of its2 FISH signal in untreated and treated ES cells. Quantification of the its2/its1 intensity ratio relative to untreated cells is shown. Bars are means (SD) of n = 3 per condition from one out of three independent experiments. *, P < 0.05. (G) Control (Rosa26CreERT2/+; NleFlox/+) ES cells were treated or not with 4-OHT. At day 3, FISH experiments using its2 probe and PCR analysis were performed showing that in the presence of a remaining functional Nle allele, large ribosomal subunit maturation was not affected.

Nle inactivation induces defects in rRNA maturation and accumulation of the large preribosomal subunit. (A) Schematic representation of the two major pre-rRNA processing pathways in mammalian cells. Localization of probes is indicated under the 47S primary transcript. Predictions based on the known function of NLE orthologue in yeast of rRNA maturation defects in Nle-deficient mouse ES cells are shown in red. If this function is conserved, Nle inactivation should induce a block in the last steps of maturation of the large preribosomal subunit, leading to an accumulation in the nucleolus of the 32S and 12S pre-rRNA species and the decreased export in the cytoplasm of mature 28S and 5.8S rRNA. 32S and 12S pre-rRNA can be detected by Northern blot or FISH analysis using probes directed against the pre-rRNA internal sequence its2 (blue). Maturation of the small subunit should not be directly affected. Thereby, the levels of the corresponding pre-rRNA detected with an its1 probe (green) should remain unchanged. Similarly, levels of 47S primary rRNA transcript detected with a 5′ets probe (purple) should not be directly affected. (B–D) Northern blot analysis of total RNA from untreated or treated ES cells. (B) Quantification of 12S/5.8S, 32S/28S, and 30S/18S intensity ratios. Northern blots analysis with 28S, 18S, 5.8S, or its2 probe was obtained at least twice. (E–G) FISH experiment on untreated and treated ES cells. (E) Representative images of two independent FISH experiments with its2, its1, or 5′ets performed on ES cells untreated or treated with 4-OHT at day 3. Cells were labeled with Hoechst. Bar, 5 µm. (F) Representative FACS profiles of its2 FISH signal in untreated and treated ES cells. Quantification of the its2/its1 intensity ratio relative to untreated cells is shown. Bars are means (SD) of n = 3 per condition from one out of three independent experiments. *, P < 0.05. (G) Control (Rosa26CreERT2/+; NleFlox/+) ES cells were treated or not with 4-OHT. At day 3, FISH experiments using its2 probe and PCR analysis were performed showing that in the presence of a remaining functional Nle allele, large ribosomal subunit maturation was not affected.

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