4606 Nucleic Acids Research, 2006, Vol. 34, No. 16 Figure 7. Determination of NOP132-interacting region of DDX47. (a) Summary of the region of NOP132 that associates with DDX47 and DDX18. The three coiled-coil structures are shown as black bars. (b) Deletion constructs of NOP132 in the vector pACT2 were examined for their interaction with DDX47 or DDX18 in the vector pAS1 using a yeast two-hybrid assay. Right panels show the results of the b-galactosidase filter assay and colony growth assay. b-gal activity indicates that an interaction had occurred. Abbreviations: + yeast two-hybrid interaction between NOP132 deletion construct and DDX47 or DDX18; +/ weak interaction; no interaction. (c) Sequence alignment of the C terminus of DDX18 and DDX47 showing 37.3% identity between these proteins. Two dots indicate identity and a single dot indicates chemically similar amino acids. in transcription (43), and is essential for growth (44). Because both Has1 and Rrp3 are essential for growth and are involved in rRNA production, DDX18 and DDX47 might have similar functions in mammalian cells. We observed that FLAG-tagged DDX47 interacts with 30S and 32S pre- rRNAs, suggesting that DDX47 is involved in early steps in pre-rRNA processing. It is likely that a main role of DDX47 could be in 60S ribosome subunit assembly, because DDX47 associates more efﬁciently with pre-rRNAs from 26S to 5.8S pathway as shown in Figure 4c. RNase treatment released most of the proteins of the NOP132-, DDX47-, and DDX18-associated complexes, dem- onstrating that NOP132-, DDX47-, and DDX18-associated complexes contain RNAs, which could act as assembly fac- tors. Because DDX47 and DDX18 belong to the DEAD- box RNA helicase family, it is likely that these proteins directly bind to RNAs that link to other proteins. NOP132 also has an RNA binding motif in its amino terminal region that can bind RNAs in vitro (T.Sekiguchi et al. unpublished data) and thus may directly bind to RNAs. Association of NOP132 with DDX47 or DDX18 was also dependent on RNA, as shown in Figure 2a, although we cannot rule out the possibility that NOP132 directly associates with either DDX47 or DDX18. A possible role for NOP132 in proper nucleolus localization of DDX47 during ribosome biogenesis It is thought that within the nucleolus, there are subcompart- ments for vectorial maturation of pre-ribosomes, with the transcription of the rDNA occurring at the interface between the FCs, where nascent transcripts reach out into the body of the DFC into the GC, as pre-rRNA processing occurs [reviewed in (45)]. With downregulation of NOP132, DDX47 appeared to be localized to the GC, but not to either the FC or DFC, suggesting that DDX47 is localized at the nucleolar periphery (probably the GC region) in the absence of NOP132 and that DDX47 might be retained in the FC