A subset of SSU components is required for A′ cleavage. (A) HeLa cells were transfected with control siRNAs or those targeting SSU processome components. For UTP12, IMP3, IMP4, and UTP23, mRNA levels (normalized to GAPDH) were determined by qPCR and are given graphically. For UTP10 and MPP10, proteins were extracted and analyzed by Western blotting. We have previously reported the effectiveness of the other knockdowns (Sloan et al. 2013b). (B) RNA was extracted from RNAi-treated cells, separated by agarose-glyoxal gel electrophoresis, and analyzed by Northern blotting using probes hybridizing in the 5′ETS or ITS1 as indicated. Mature rRNAs were visualized by methylene blue staining (MB). (C) HeLa cells depleted of various SSU processome components were pulse-labeled, and RNA was analyzed by agarose-glyoxal gel electrophoresis and visualized using a phosphorimager.
