or DFC in the nucleolus by NOP132 during ribosome bio- genesis. Consistently, FLAG-tagged DDX47 associated with 30S and 32S pre-rRNAs in the cell, indicating that DDX47 has a role in primary pre-rRNA processing. It has been reported that NOP132/NOL8 is upregulated in diffuse-type gastric cancer and that downregulation of NOP132 induces apoptosis (19), suggesting that NOP132 is involved in carcinogenesis. We found that p53 co- precipitated with FLAG-NOP132. Although the association of NOP132 with p53 is suggestive, it is possible that over- expression of NOP132 is a consequence rather than an effector of carcinogenesis. It was also reported that NOP132/NOL8 is a phospho- protein (19). Consistently, NOP132 associated with several kinases that are involved in signal transduction (Table 1). Thus, these kinases might phosphorylate NOP132 and inﬂu- ence its function. It is also possible that NOP132 regulates DDX47 helicase activity either positively or negatively by associating with these kinases. Such is the case for telomere repeat binding factor (TRF) proteins and Bloom syndrome (BLM) helicase; the interaction of TRF1 and TRF2 with BLM has been reported to stimulate the BLM helicase activity (46,47). The structure of the DEAD-box RNA helicases indicates that the HELICc domain is located downstream of the heli- case domain (DEADc). In the HELICc domain, there are three conserved DEAD-box RNA helicase motifs, IV, V, and VI, within the HELICc domain (Figure 6c), that possibly function in substrate and g phosphate binding (48). The region containing the HELICc motif of DDX47 interacted directly with the coiled-coil motif of NOP132 and was responsible for the nucleolar localization of the DDX47- EGFP fusion protein (Figure 6). Because DDX18 has a high level of amino acid identity with DDX47, especially in the HELICc motif, it is possible that both proteins interact with NOP132 through this motif. Although another DEAD- box RNA helicase, DDX21, was also found in the NOP132 complex (Figure 1a), direct interaction between DDX21 and NOP132 was not detected by yeast two-hybrid analysis (data not shown), suggesting that DDX21 binds to the NOP132 complex through interactions with other proteins or RNAs. We previously reported that NOP132 could be the human counterpart of yeast Nop8 (15). Because Nop8-depleted cells contain reduced levels of free 60S ribosomes and polysomes and accumulate half-mer polysomes, Nop8 is probably involved in 60S ribosome biogenesis (16). Recently, it was reported that yeast Nop8 functionally interacts with DEAD- box RNA helicase Dbp6, which also interacts with Rpl3 and Rsa3, as shown by synthetic lethal mutant screening, and is involved in 60S ribosomal subunit formation (49). Dbp6 interacts with another DEAD-box RNA helicase, Dbp9, which is also involved in 60S ribosome biogenesis (50). A region of high similarity in amino acid sequences between NOP132 and Nop8 encompasses the DDX47- interacting region of NOP132, suggesting functional con- servation between the two proteins and the involvement of NOP132 in 60S ribosome biogenesis. With regard to this possibility, we demonstrated that NOP132 associates with a novel nucleolar protein, NOP254/NNP72 (35), which is a human ortholog of yeast Npa1, which is a component of Nucleic Acids Research, 2006, Vol. 34, No. 16 4607 very early pre-60S ribosomal particles. Consistently, Npa1 is shown to associates with Nop8 and other nucleolar proteins (31,51). Together these data suggest that NOP132 participates in various stages of human ribosome biogenesis. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Drs H. Kobayashi, H. Nishitani, K. Mihara, and N. Ishihara for helpful discussion and support. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (T.S.), Grants-in-Aid for Specially Promoted Research (T.N.), and a Pioneer Research on Genome the Frontier, Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Science Research, from the Japan Society for the Promotion of Science (N.T.). Funding to pay the Open Access publication charges for this article was provided by Kyushu University. Conflict of interest statement. None declared. REFERENCES 1. Granneman,S. and Baserga,S.J. (2004) Ribosome biogenesis: of knobs and RNA processing. Exp. Cell Res., 296, 43–50. 2. Fromont-Racine,M., Senger,B., Saveanu,C. and Fasiolo,F. (2003) Ribosome assembly in eukaryotes. Gene, 313, 17–42. 3. Venema,J. and Tollervey,D. (1999) Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet., 33, 261–311. 4. Takahashi,N., Yanagida,M., Fujiyama,S., Hayano,T. and Isobe,T. 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