4602 Nucleic Acids Research, 2006, Vol. 34, No. 16 ( d ) Figure 4. Association of DDX47 with pre-rRNAs. (a) HeLa cells were transiently transfected with two dsRNAs for each RNAi experiment as described in the Materials and Methods. WT, Control fruit fly luciferase oligonucleotide; DDX47 RNAi, oligonucleotides DDX47-1 and -2; NOP132 RNAi, oligonucleotides NOP132-B and -C. After transfection for 1 d, total RNAs were purified and processed for Northern blot analysis using ITS1, ITS2, 28S, 18S, 5.8S, or a-actin DNAs as probes (shown in parentheses). RNA from control (WT), NOP132 RNAi-treated, and DDX47 RNAi-treated cells was electrophoresed on a formaldehyde agarose gel as described in the Materials and Methods. Radioactivity in each lane was measured using the Fuji BAS2500 Image Analyzer (Fuji Photo Film Co. Ltd, Japan). Relative radioactivity of each rRNA or rRNA precursor is shown at the bottom of the panels. (b) Proteins from the siRNA-treated cells were isolated and processed for western blotting analysis using anti-NOP132N, anti-DDX47, or anti-RAN as a loading control. (c) 293 cells were transiently transfected with FLAG- DDX47. After transfection for 2 d, a cell lysate was prepared and the DDX47-associating complex was purified with anti-FLAG. RNA was purified from the immunoprecipitate and processed for Northern blot analysis using ITS1, ITS2, 28S, 5.8S, or 18S oligonucleotide probes. As a control, an untransfected 293 cell lysate was prepared and processed as above. Relative radioactivity of each rRNA or rRNA precursor is shown at the bottom of the panels. (d) Schematic representation of the pre-rRNAs detected in the Northern blots. Possible points where DDX47 might act are indicated in the figure. The pre-rRNA cleavage sites (1- 3, 30, 40) are indicated by arrowheads. deduced from the known functions of their yeast orthologs. We also examined the association of FLAG-tagged NOP132 with the rRNA precursors; however, we did not detect a signiﬁcant amount of rRNA precursor (data not shown). This might be the result of a transient association of NOP132 with pre-rRNAs. As a control, we analyzed a non-tagged control strain in parallel in Figure 4c. Proper nucleolus localization of DDX47 by NOP132 Inhibition of rRNA synthesis by actinomycin D results in re-localization of DDX47 from the nucleolus to the entire nucleus, along with other nucleolar proteins (37). Although DDX47 is localized exclusively in the nucleolus in quiescent cells, an anchoring protein for DDX47 in the nucleolus has not been identiﬁed. We examined the possibility that