Published online 8 September 2006 Nucleic Acids Research, 2006, Vol. 34, No. 16 4593–4608 doi:10.1093/nar/gkl603 NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47 1 1 Takeshi Sekiguchi*, Toshiya Hayano , Mitsuaki Yanagida , Nobuhiro Takahashi1 and Takeharu Nishimoto Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan and 1Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, fuchu, Tokyo 183-8509, Japan Received April 5, 2006; Revised July 11, 2006; Accepted August 3, 2006 ABSTRACT Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP bind- ing protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabo- lism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA pre- cursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus. INTRODUCTION Ribosome biogenesis is a highly complex and coordinated process that occurs not only in the nucleolus but also in the nucleoplasm and cytoplasm (1) that has been studied exten- sively in yeast systems using both biochemical and genetic analyses (2). Based on the protein composition of several yeast ribosomal subunit precursors recently characterized by proteomic approaches, more than 170 factors are predicted to participate in ribosome biogenesis [see review in (3)]. Thus, proteomic approaches have provided additional evidence for a ribosome assembly pathway in eukaryotes and have conﬁrmed the dynamics of the entire process (4). Ran is a member of the Ras superfamily of small G pro- teins and a nuclear protein with several functions, including nucleocytoplasmic transport of many types of proteins and of nucleic acids across nuclear membranes and ribosome bio- genesis through Dis3, an exosome subunit (5–9). The gene encoding the yeast homolog of RCC1 (SRM1), the guanine nucleotide exchange factor for Ran, genetically interacts with GTR1 in yeast (10). Gtr1 and Gtr2 are associated with each other and belong to a novel subfamily of small G pro- teins (11) and play various roles in cellular metabolism (12,13). Gtr1 is associated with Rpc19 (14) and Nop8 (15) which is an essential nucleolar protein required for 60S ribo- some biogenesis (16) and is associated with nucleolar protein Nip7 (17), suggesting that Gtr1 could play roles in multiple steps of ribosome biogenesis. Human RRAG A/Rag A/FIP-1 is a functional homolog of yeast Gtr1 (18) and is associated with an essential nucleolar protein, NOP132, which interacts with RRAG C, RRAG D, and human NIP7 and might anchor these proteins to the nucleolus (15). Amino acid sequence comparison revealed that NOP132 is similar to yeast Nop8. Thus, NOP132 might have a role in ribosome biogenesis. NOP132 was found to be upregulated in diffuse-type gastric cancer in an attempt to identify novel therapeutic targets for this disease (19). RNA helicases from the DEAD-box family are present in almost all organisms and have important roles in RNA metabolism. They participate in many processes, such as the synthesis, processing, export, and degradation of RNA [see review in (20)]. In humans, 36 members of the DEAD-box family of putative RNA helicases have been identiﬁed and are hypothesized to have roles in differentia- tion and carcinogenesis (21). Saccharomyces cerevisiae has *To whom correspondence should be addressed. Tel: +81 92 642 6177; Fax: +81 92 642 6183; Email: sekigu@molbiol.med.kyushu-u.ac.jp Present addresses: Toshiya Hayano, Department of Bioscience and Bioinformatics, College of Information Science and Engineering, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu 525-8577, Japan Mitsuaki Yanagida, Institute for Environmental and Gender Specific Medicine, Juntendo University, Graduate School of Medicine, 2-1-1, Tomioka, Urayasu, Chiba 279-0021, Japan 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.