in hamster cells. Each of the four proteins was fused with EGFP and transfected with DsRed-NOP132 into BHK21 cells. Fluorescence microscopy examination revealed that these four proteins co-localized with NOP132 in the nucleo- lus (Figure 3a). Consistent with this ﬁnding, DDX47 localizes to the nucleolus under normal growth conditions (34). Surprisingly, NOP132 co-localized with EGFP-RPL3, which was expected to be localized mainly in the cytoplasm. However, Andersen et al. (35) reported that RPL3 was detected in a proteomic analysis of the human nucleolus. Thus, it is likely that NOP132 co-localized with a nucleolar fraction of RPL3. We also observed that DDX18 and KIAA0539 localized to the nucleolus (Figure 3a and b). We believe that KIAA0539, which has some similarity to yeast Urb1p/Npa1, is a novel nucleolar protein (Figure 3a), and therefore named it NOP254, based on its nucleolar localization and its molecular weight of 254 kDa. In addition, we conﬁrmed the nucleolar localization of endogenous DDX47 and DDX18 by immunostaining using afﬁnity puri- ﬁed polyclonal antibodies against each protein (Figure 3b). Roles of NOP132 and DDX47 in pre-rRNA processing To examine the roles of NOP132 and its associated proteins in pre-rRNA processing, RNAi experiments of NOP132 and DDX47 were carried out. After introduction of the siRNAs to the cells, total RNA was puriﬁed and used for northern blotting analysis (Figure 4a). RNAs from same number of cells (about 4000 cells) were loaded onto the gel to compare the amount of rRNAs and rRNA precursors in wild type, NOP132 RNAi-treated and DDX47 RNAi-treated cells. Downregulation of NOP132 slightly decreased 18S rRNA and rRNA precursors, including 45S/47S rRNA, suggesting that NOP132 have a role in rRNA processing. Likewise, downregulation of DDX47 also decreased the amounts of mature rRNAs and rRNA precursors in HeLa cells, suggest- ing that DDX47 is also involved in rRNA production. We cannot rule out possibility that some pre-rRNAs are degraded. We observed that the total amount of RNA per cell decreased on average about 30% in NOP132-downregulated cells and about 40% in DDX47-downregulated cells in three separate cell samples. It could be possible that this decrease in RNA was caused by a kind of stringent control [reviewed in (36)], which might be induced by interruption of rRNA processing by downregulation of NOP132 or DDX47. Figure 4b shows that addition of NOP132 siRNA and DDX47 siRNA down- regulated expression of their respective proteins. The amount of actin mRNA was also decreased in these cells (Figure 4a, lowest panel), suggesting that DDX47 might function in mRNA splicing. Consistently, many proteins involved in mRNA splicing were found in DDX47-associated complexes (Table 1). Next, we examined the association of DDX47 with rRNA precursors by transfecting 293 cells with FLAG-tagged DDX47. RNAs from the FLAG-DDX47-expressing cells were puriﬁed by immunoprecipitation with anti-FLAG and used for northern blotting analysis (Figure 4c). FLAG- DDX47 associated with 32S, 30S, 26S, 17S rRNA precursors and 18S, 5.8S and 28S rRNAs, suggesting that DDX47 is involved in early stages of pre-rRNA processing, probably after the primary rRNA transcript is synthesized (Figure 4d). Because DDX47 associated with both 30S and 32S rRNA Nucleic Acids Research, 2006, Vol. 34, No. 16 4601 Figure 3. Co-localization of NOP132 with DDX47, DDX18, RPL3, and NOP254 in the nucleolus. (a) BHK21 cells were transiently cotransfected with DsRed-NOP132 (red) and DDX47-EGFP, DDX18-EGFP, RPL3-EGFP, or KIAA0539/NOP254-EGFP (green). The cells were fixed and processed for confocal microscopy imaging as described in the Materials and Methods. Scale bars, 10 mm. (b) HeLa cells were fixed and immunostained with affinity purified rabbit polyclonal anti-DDX18 or anti-DDX47 followed by fluorescein-isothiocyanate-conjugated anti-rabbit secondary antibody. DNA was stained with Hoechst dye. Phase images are shown in the right panels. precursors, DDX47 might be involved in 18S and 28S rRNA maturation. Consistent with this, DDX47 associated with both 18S and 28S rRNA maturation proteins, as shown in Table 2, in which the functions of human proteins were