Sf9 cells with recombinant baculoviruses encoding NOP132 as described previously (15). Brieﬂy, Sf9 cells were cultured in 250 ml disposable Erlenmeyer ﬂasks at 27 C under rota- tion at 125 r.p.m. in Grace’s insect medium (Invitrogen) sup- plemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were maintained at a density of 0.5–6.0 · 106 cells/ml. Cells were seeded at 3.0 · 106 cells/ml and infected with a 1:100 dilution of baculovirus stocks, and cultured for 48–72 h. The cells were collected and lysed with the immunoprecipitation buffer [50 mM Tris–HCl, pH 7.4; 1 mM EDTA; 150 mM NaCl; 0.1% (v/v) Nonidet P40; 1 mM phenylmethyl sulfonyl ﬂuo- ride; 0.1 mg/ml aprotinin; and 1 mM dithiothreitol (DTT)]. The extract was obtained by centrifugation at 13 000 g for 10 min at 4 C. The extract was diluted to 500 ml with immunoprecipitation buffer. Twenty microgram of either GST, GST-DDX47, GST-DDX18, or GST-GRWD1 bound to the glutathione-Sepharose-4B beads was mixed with baculovirus-produced NOP132. After incubation at 4 C for 30 min, the beads were pelleted, washed four times with immunoprecipitation buffer, and suspended in 50 ml of SDS–PAGE sample buffer (62.5 mM Tris–HCl, pH 6.8; 100 mM DTT; 2% (w/v) SDS; and 10% glycerol). Bound proteins were subjected to SDS–PAGE and NOP132 was detected using anti-NOP132N. Immunoblotting and antibodies Protein samples were subjected to SDS–PAGE using 5–20% gradient polyacrylamide gels (PAGEL, ATTO, Japan) and the protein bands were analyzed by immunoblotting. Immunopositive bands were visualized using the ECL kit (GE-Healthcare) as recommended by the supplier. The anti- NOP132N and anti-RRAG A antibodies were described pre- viously (15). The anti-human DDX18 and DDX47 antibodies were raised by immunizing rabbits with GST-DDX18 and GST-DDX47 proteins, respectively, and were puriﬁed by afﬁnity puriﬁcation methods using GST-fusion proteins that were transferred onto a ﬁlter. Mouse anti-RAN (Cat. no. 610 340) was purchased from BD Bioscience (San Jose, CA). Immunofluorescence Transfected cells on coverslips were ﬁxed with 1 ml 4% paraformaldehyde for 5 min and were processed for immunostaining as described (25). Cells were stained with 1 mg/ml Hoechst 33 342 and mounted with Vectashield or with Vectashield containing 40-6-Diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). A Zeiss Axiophot microscope was used for sample analysis using standard microscopic methods. Digital imaging of the stained cells was also obtained using an Olympus laser-scanning microscope LSM-GB200 system, as described (18). Yeast two-hybrid assay A yeast two-hybrid assay (26) was performed using the S.cerevisiae Y190 strain (a gal4 gal80 his3 trp1 ade2 ura3 leu2 URA3::GAL1-lacZ LYS2::GAL1-HIS3 cyhr) to test protein interactions in vivo, as described (22). S.cerevisiae cells were grown in YPD (2% glucose, 2% peptone, and 1% yeast extract) or SD-Trp-Leu-His + 3-Aminotriazol Nucleic Acids Research, 2006, Vol. 34, No. 16 4595 (2% glucose, 0.67% yeast nitrogen base without amino acids, and 25 mM 3-Aminotriazol, supplemented with all essential amino acids except for tryptophan, leucine, and his- tidine). Amino acids were added to a ﬁnal concentration of 20–50 mg/ml. The solid media contained 2% agar in addition to the components described above. The b-galactosidase chromogenic ﬁlter assays were performed by transferring the yeast colonies onto nitrocellulose ﬁlters (Protran BA85; Schleicher and Schuell, Germany). The yeast cells were par- tially lysed by submerging the ﬁlters in liquid nitrogen for 1 min. Filters were processed as described previously (15). Color, representing a positive signal, appeared within 120 min at 30 C (Figure 7). Recombinant DNA Human DDX47 cDNA was isolated by RT–PCR using the human Burkitt’s lymphoma cDNA library (Becton Dickinson, Franklin Lakes, NJ). DDX18 cDNA was purchased from Invitrogen (Carlsbad, CA). The control FLAG vector was pFLAG-CMV-5a (Sigma). Enhanced green ﬂuorescent protein (EGFP)-NOP132 and the red ﬂuorescent protein (DsRed)-NOP132 were used to visualize the subcellular localization of proteins as described (15). GST-DDX18, GST-DDX47, and GST-GRWD1 were constructed by insert- ing cDNA fragments into pGEX-KG. Deletion constructs of DDX47 and NOP132 in pAS1, pACT2, and pEGFPc1 vectors were prepared by PCR. Kiaa0539 cDNA was provided by Dr Ohara at the Kazusa DNA Research Institute (Chiba, Japan). The EcoRI–XhoI fragment of the Kiaa0539 cDNA was inserted into the EcoRI–XhoI sites of the pEGFPc2 vector. To obtain FLAG-Coil [without a nuclear localization signal (NLS)], a NOP132 cDNA fragment (nucleotide no. 2000–2870) was ampliﬁed by PCR and inserted into the EcoRI site of the pcDNA3.1 (Hygro) vector. Human ribosomal internal transcribed spacer 1 (ITS1), internal transcribed spacer 2 (ITS2), external transcribed spacer 2 (ETS2), 28S rRNA, 18S rRNA, and 5.8S rRNA were sub- cloned in the pUC118 vector. Each construct was checked by automated DNA sequencing using an ABI PRISMR 3100 sequencer (Applied Biosystems, Foster City, CA). RNAi Two pairs of small interfering RNAs (siRNAs) were chemically synthesized (Hokkaido System Science, Sapporo, Japan) and annealed before transfection as described previ- ously (27). Transfection was performed on 2 · 104 HeLa cells/20 mm tissue culture plate or 5 · 105 HeLa cells/ 90-mm tissue culture plate with a ﬁnal concentration of 200 mM siRNA duplex using Oligofectamine reagent (Invitrogen) according to the manufacturer’s instructions. Using ﬂuorescein-isothiocyanate-conjugated control oligonu- cleotide, there was nearly 100% incorporation of the oligonu- cleotide under our experimental conditions, as observed under a ﬂuorescence microscope. The oligonucleotide sequences used for NOP132 and DDX47 RNAi experiments were as follows: NOP132; mixtures of NOP132-B (50-CACUUCACCUAAUGAGCCATT-30/50-UGGCUCAUU- AGGUGAAGUGTT-30), and NOP132-C (50-CAGUUUC- CUUAGGUGAGCCTT-30/50-GGCUCACCUAAGGAAAC- UGTT-30) (15). DDX47; mixtures of DDX47-1