Box C/D snoRNP proteins are important for efficient A′ processing but essential for downstream removal of the 5′ETS. (A) Schematic outline of the pre-rRNA processing pathway in human cells. Cleavages important for 18S rRNA processing are indicated. Probes used in this study are indicated above the schematic of the 47S rRNA. (ETS) external transcribed spacer, (ITS) internal transcribed spacer. An aberrant pre-rRNA, 30SL5′, which accumulates when A′ processing is inhibited, is shown in gray within a box. (B) RNA from control cells, or those depleted of box C/D snoRNP proteins by RNAi, was separated by agarose-glyoxal gel electrophoresis and transferred to nitrocellulose membrane. Northern blotting, using probes hybridizing in the 5′ETS or ITS1, was performed and pre-rRNAs detected using a phosphorimager. Mature rRNAs were visualized using methylene blue staining (MB). (C) HeLa cells were transfected with either control siRNAs or those targeting box C/D snoRNP proteins, and 48 h later, cells were pulse-labeled using 32P orthophosphate. RNA was extracted, separated by agarose-glyoxal gel electrophoresis, and visualized using a phosphorimager. Total rRNA (28S/18S) was visualized by ethidium bromide staining (UV).
