Fig 1: EMG1 is part of a pre-ribosomal subcomplex containing NOP14, NOC4L and UTP14a. (A) His-ZZ-tagged EMG1 was immobilised on IgG sepharose and after incubation with HeLa cell lysate, complexes were treated with RNase (+) or left untreated (-). EMG1 and co-precipitated proteins were eluted by cleavage using TEV protease and eluates were analysed by western blotting using antibodies against the indicated endogenous proteins. (B) HeLa cells were transfected with non-target siRNAs (siNT) or siRNAs against NOP14, NOC4L, UTP14A or EMG1 and harvested after 72 h. Proteins were analysed by western blotting using antibodies against the indicated ribosome biogenesis cofactors, and tubulin as loading control. (C) HeLa cells were treated with siRNAs as in (B), total RNA was extracted and pre-rRNA levels were monitored by northern blotting using a probe hybridising the 5’ end of ITS1. The mature 18S and 28S rRNA, visualised by methylene blue staining, served as a loading control. (D) HeLa cells were transfected with siRNAs as in (B) and after 72 h, cells were pulse-labelled with 32P-orthophosphate and then grown in normal media for a further 3 h. Total RNA was extracted, separated by agarose-glyoxal gel electrophoresis and newly synthesised precursor and mature rRNAs were detected using a phosphorimager. Mature 28S rRNA, visualised by ethidium bromide staining, served as a loading control. (E) The ratio of newly synthesised 18S to 28S rRNAs was quantified in the pulse-labelling experiments and data from three independent experiments are shown as mean +/- SEM.