Figure 6.

Human NOP2/NUSN1 is required for 60S ribosomal subunit biogenesis. (A) HCT116 cells were infected with doxycycline-inducible NOP2 shRNA expressing lentivirus. After puromycin selection, cells were induced with 200 ng/ml doxycycline (Dox+) for four days. Non-induced (Dox–) cells were used as control. NOP2/NSUN1 depletion efficiency was determined by western blot. (B) A fraction of cells from (A) was analyzed by polysome profiling using total cell lysates. Dox+ and Dox– indicate doxycycline-induced and non-induced control, respectively. (C) HCT116 cells were transfected with non-targeting control (siC) or NOP2 siRNA. 72 h after transfection, cells were treated with 5 μg/ml puromycin for the indicated time. Puromycylation of nascent peptides and NOP2/NSUN1 depletion efficiency, was determined by Western blot using puromycin and NOP2 antibodies. (D) Densitometry quantification of the puromycin signal from each lane from (C) was normalized to the corresponding GAPDH signal. The data is presented as the mean of two independent biological replicates ± standard deviation (SD). Statistical significance between NOP2 depleted samples and non-targeting control samples was calculated using a two-tailed independent Student's t-test. (E) High molecular weight complexes containing pre-rRNAs and tightly associated ribosome assembly factors, referred to as ‘core’ preribosomal particles isolated from HCT116 nuclear extracts under high-salt conditions were separated on sucrose gradient ultra-centrifugation followed by fractionation. RNA and proteins were isolated from each fraction and analyzed by western blotting (WB) or northern blot (NB) with the indicated antibodies or probe, respectively. (F) Nuclear extract from HCT116 cells was separated on sucrose gradient ultra-centrifugation followed by fractionation. Proteins were extracted from each fraction and analyzed by western blot with the indicated antibodies.