Figure 5.

Human NOP2/NSUN1 is required for efficient pre-rRNA processing. (A) Schematic overview of human precursor rRNA processing and location of probes used for northern blot indicated in colored areas (lavender, yellow, green and blue). (B) HCT116 cells expressing empty vector (Vector), siRNA resistant NOP2/NSUN1 WT, or the C513A catalytically inactive mutant were transfected with non-targeting control (siC) or NOP2 siRNA #2. After 72 h, total RNA was separated on formaldehyde denaturing agarose gel and analyzed by Northern blot using 5′ETS, 3′ETS, ITS-1, ITS-2, 18S, 28S and 7SL probes. A fraction of cells was collected to determine endogenous NOP2/NSUN1 depletion and ectopic expression of NOP2/NSUN1 WT or C513A by Western blot using a NOP2 antibody. (C) Densitometry quantification of each rRNA precursor from (B) normalized to 7SL RNA. The data are presented as the mean of three independent biological replicates ± standard deviation (SD). P-values comparing empty vector with NOP2/NSUN1 knockdown (siNOP2#2 + Vector) were calculated using a two-tailed independent student t-test.