NOP132 acts as a nucleolar anchoring protein for DDX47. Reduction of NOP132 expression by siRNA caused DDX47 to localize to the nucleolar periphery, probably in the granular component (GC) region (Figure 5a). This result suggests that DDX47 can still be recruited to the nucleolus in the absence of NOP132 but that NOP132 is required for retention of DDX47 in the ﬁbrillar center (FC) and/or dense ﬁbrillar com- ponents (DFC) region of the nucleolus. When actinomycin D was applied to HeLa cells for 8 h, DDX47 localized uni- formly throughout the nucleus (Figure 5b), suggesting that recruitment and retention of DDX47 to the nucleolus requires active transcription (Figure 5c). To conﬁrm that NOP132 plays a role in binding and recruiting DDX47 to the FC or DFC region of the nucleolus, we used a FLAG-tagged NOP132 coiled-coil domain fragment (FLAG-Coil), which contains the minimum region required to interact with DDX47 (see Figure 7) but lacks the NLS; this form of NOP132 localized mainly in the cytoplasm (Figure 5e). When EGFP-DDX47 was expressed alone in 293 cells, most of the EGFP-DDX47 localized to the nucleolus (Figure 5f). When FLAG-Coil was transiently cotransfected with EGFP-DDX47 into 293 cells, EGFP-DDX47 distributed evenly in the nucleus and FLAG-Coil was distributed in both the cytoplasm and the nucleus (Figure 5d, upper and lower panels). In some cells in which FLAG-Coil expression was low and DDX47 expression was high, the FLAG-Coil fusion mutant NOP132 protein was localized in the nucleus (Figure 5d, middle panels). Thus, it appears that the sub- cellular localization of EGFP-DDX47 was inﬂuenced by the relative expression levels of the FLAG-Coil protein and EGFP-DDX47. Together, these results suggest that NOP132 is required for DDX47 to localize in the nucleolar-organizing region during ribosome biogenesis. Next, we identiﬁed the DDX47 region responsible for sub- cellular localization using a series of EGFP-fused DDX47 deletion proteins in BHK21 cells (Figure 6a and b). Nuclear export or cytoplasmic retention signals were found to reside between amino acid residues 50 and 100. A NLS in the C-terminal region was found to reside between residues 400 and 456. A nucleolar localization region was predicted to be between amino acid residue 200 and the C terminus because the EGFP fusion protein containing the region from residue 200 to the C terminus had a nucleolar staining pattern but the EGFP fusion protein containing the region from residue 300 to the C terminus had lost its nucleolar localization (Figure 6a). A search for known protein sequence motifs was performed with the Conserved Domain Database (CDD) (38); it revealed that the region from residue 200 to the C terminus of DDX47 contains the helicase superfamily C-terminal domain (HELICc) motif, which is found in a wide variety of helicases and helicase-related proteins. To examine whether the region that determines the nucleolar localization of DDX47 is the region that interacts with NOP132, we per- formed a yeast two-hybrid assay using DDX47 as the bait and found that the same region of DDX47, residue 200 to the C terminus, was also responsible for binding to NOP132 (Figure 6a and c). Thus, the NOP132-interacting region of DDX47 also functions as a nucleolar localization signal. This result is consistent with the idea that NOP132 is required for the proper nucleolar localization of DDX47. In contrast, NOP132 siRNAs did not affect the nucleolar Nucleic Acids Research, 2006, Vol. 34, No. 16 4603 localization of DDX18 (data not shown), suggesting that pro- teins other than NOP132 determine the nucleolar localization of DDX18. NOP132 coiled-coil structure is responsible for binding to both DDX18 and DDX47 We used a yeast two-hybrid assay to further examine the region of NOP132 responsible for binding to DDX18 and DDX47 (Figure 7a and b). Serial deletion constructs of NOP132 were constructed in the vector pACT2 (Figure 7b) and subjected to b-galactosidase ﬁlter assays using DDX47 or DDX18 as bait. The results indicated that the region of NOP132 containing two coiled-coil structures was responsi- ble for binding to both of the DDX proteins. The coiled-coil structure is known to mediate protein–protein interactions and subunit oligomerization [see review in (39)]. Because the region of DDX47 that interacts with NOP132 contains a HELICc motif (as described above), it is likely that the NOP132 coiled-coil motif interacts directly with the DDX47 HELICc motif. DDX18 also contains a HELICc motif at the C terminus, has high sequence similarity to DDX47 in that region, and interacts with NOP132 (Figure 7c). The HELICc motif of DDX18 might also directly interact with the coiled- coil motif of NOP132. Because other DDX family proteins have an amino acid sequence similar to the HELICc motif, we examined the possibility that the other DDX family pro- teins also bind to NOP132. We performed yeast two-hybrid assays on DDX3, DDX5, DDX21, and DDX49; however, none of these DDX proteins interacted with NOP132 (data not shown), suggesting that the interaction with NOP132 is speciﬁc to DDX47 and DDX18. DISCUSSION NOP132 and DDX47 are involved in human ribosome biogenesis The present study demonstrates that NOP132 physically inter- acts and co-localizes with DDX47 and DDX18. Their associ- ated complexes contained a number of proteins found in previously reported human pre-rRNP complexes (4,30–32). We also found that FLAG-tagged DDX47 associated with pre-rRNA precursors and RNAi-mediated downregulation of NOP132 resulted in mislocalization of DDX47. These results, coupled with the nucleolar localization of NOP132 and DDX47, strongly suggest that NOP132 and DDX47 are involved in human ribosome biogenesis cooperatively. The yeast ortholog of human DDX47 is Rrp3 (identity, 57%), which plays a role in the cleavage of A0 (the 50 exter- nal transcribed spacer), A1 (the 50 end of the 18S rRNA) and A2 (located 1.9 kb away in ITS1) and in the processing of pre-rRNA, and is required for 18S rRNA production in yeast cells (40). Although it was reported that the yeast ortholog of human DDX47 is Prp3 with 56% identity (21), we did not ﬁnd such identity between these proteins. DDX18 also potentially has several roles in early ribosome biogenesis: it is similar to yeast Has1 (identity, 60%), which is a component of 90S and pre-60S ribosomal particles (41), is an essential trans-acting factor involved in 40S ribo- somal subunit biogenesis (42), is a direct target of MYC