Figure 6.

Knockdown of RPL5 or RPL11 retains nucleolar RNA by preventing its export. (A) Knockdown of RPL5 or RPL11 retained RNA content in the nucleolus. The total RNA was isolated from the isolated nucleoli of MCF-7 cells transfected with the indicated siRNAs for 48 h. The total RNA (left) and 28S rRNA (right) levels were quantified by spectrophotometry and RT–qPCR, respectively. The level of the siCont-treated cells was normalized to 1.0. (B) Downregulation of MYBBP1A, RPL5, or RPL11 did not affect the transcription levels of pre-rRNA that had been reduced by TIF-IA knockdown. Nuclear run-on assays were performed to measure the transcription levels of pre-rRNA in MCF-7 cells transfected with the indicated siRNAs for 48 h. Transcription from the rRNA gene was measured by hybridization of in vitro-synthesized ³²P-labelled run-on transcripts to immobilized plasmids containing no insert (Control) or cDNA corresponding to 28S rRNA (pre-rRNA). The assays were performed in duplicate. (C) Knockdown of RPL5 or RPL11 prevents nucleolar RNA export from the nucleolus. (Left) MCF-7 cells were transfected with the indicated siRNAs for 48 h. The RNA polymerase II was inhibited by α-amanitin and the newly synthesized rRNA in the cells was pulse labelled with BrUTP. These cells were chased in the BrUTP-free medium for 0 or 60 min, fixed, and stained with anti-BrdU (green), anti-NPM (red) antibodies, or DAPI (blue). (Right) The percentage of cells that showed BrUTP staining in the nucleoplasm at 0 or 60 min after chase. Values are given as the mean±s.d. for triplicate experiments.