4594 Nucleic Acids Research, 2006, Vol. 34, No. 16 26 DEAD-box proteins, many of which are involved in ribosome biogenesis (20). Here we used a proteomic approach to elucidate the func- tion of NOP132 in human cells. We identiﬁed NOP132- associated proteins by mass spectrometric analysis; among the identiﬁed proteins were the nucleolar DEAD-box RNA helicases DDX47 and DDX18. We also showed that DDX47-associated proteins are very similar to those associ- ated with NOP132. Because FLAG-tagged DDX47 could be co-precipitated with rRNA precursors, DDX47 may be involved in pre-rRNA processing. To understand the signiﬁcance of the association of NOP132 with DDX47, we examined the subcellular localization of DDX47 upon downregulation of NOP132 by RNA interference (RNAi). NOP132 downregulation caused mislocalization of DDX47. Herein we propose that NOP132 recruits DDX47 protein to the center of the nucleolus, where it functions in the process- ing of primary rRNA transcripts. MATERIALS AND METHODS Cell culture and transient transfection BHK21, 293, 293EBNA, and HeLa cells were grown at 37.5 C in Dulbecco’s modiﬁed Eagle medium containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) in an incubator under 10% CO2 or, for 293EBNA cells, 5% CO2. Cells were washed with TD buffer (25 mM Tris-HCl, pH 7.4; 136.8 mM NaCl; 5 mM KCl; and 0.7 mM Na2HPO4). BHK21 and 293 cells (1 · 105 cells) were transfected with 1 mg vector and 7 ml LipofectAMINETM Reagent (Life Technologies, Rockville, MD) per 35 mm dish for immunoﬂuorescence experiments. For FLAG-tagged protein co-precipitation experiments, 293EBNA and 293 cells (5 · 105 cells) were transfected with 5 mg vector and 30 ml LipofectAMINETM Reagent per 100 mm dish for 4 h in the absence of serum and antibiotics, as recommended by the supplier, and then cells were incubated at 37.5 C as pre- viously described (22). Immunoprecipitation and protein identification by peptide mass fingerprinting At 48 h post-transfection, 293EBNA cells were harvested and washed with phosphate-buffered saline (PBS) and lysed on ice for 30 min in lysis buffer (50 mM Tris–HCl, pH 8.0; 150 mM NaCl; and 0.5% (w/v) IGEPAL CA630) containing a protease inhibitor mixture. One sample (Figure 1, lane 3) was sonicated on ice for 1 s (Sonicator ; Heat System- Ultrasonics Inc., Farmingdale, NY), with a microtip, 40% cycle, and output control of 4. The soluble fraction was obtained by centrifugation at 15 000 rpm for 30 min at 4 C and incubated with 20 ml anti-FLAG M2-agarose beads (Sigma-Aldrich, St Louis, MO) for 4 h at 4 C to immunopre- cipitate NOP132-associated complexes. After the beads were washed ﬁve times with lysis buffer and once with 50 mM Tris–HCl, pH 8.0/150 mM NaCl, complexes bound to the agarose beads were eluted with 20 ml 50 mM Tris–HCl, pH 8.0/150 mM NaCl containing 500 mg/ml FLAG peptide. The eluted complexes were analyzed by SDS–PAGE. Protein-containing SDS–PAGE gel fragments were subjected to in-gel digestion with trypsin as previously described (23). The resulting peptides were recovered and analyzed for peptide mass ﬁngerprinting using a PE Biosystems Voyager DE-STR (Perkin-Elmer, Inc., Wellesley, MA) as described previously (23). Peptide masses were searched with 50 p.p.m. mass accuracy using the database ﬁtting program MS-Fit (available at prospector.ucsf.edu), and protein identiﬁcation was performed according to the criteria described previously (23). When identiﬁcation was unsuccessful using this method, the digested sample was analyzed by the LC-MS/MS method described below. Protein Identification by LC-MS/MS Analysis NOP132-associated complexes were digested with endopro- teinase Lys-C, and the resulting peptides were analyzed using a nanoscale LC-MS/MS system as described (24). The pep- tide mixture was applied to a Mightysil-RP-18 (3 mm particle; Kanto Chemical, Osaka, Japan) fritless column (45 mm · 0.150 mm, i.d.) and separated using a 0–40% acetonitrile gra- dient containing 0.1% formic acid over 80 min at a ﬂow rate of 50 or 25 nl/min. Eluted peptides were sprayed directly into a quadrupole time-of-ﬂight hybrid mass spectrometer (Q-ToF 2; Micromass, Wythenshawe, UK). MS/MS spectra were acquired by data-dependent collision-induced dissociation, and MS/MS data were analyzed using MASCOT software (Matrix Science, London, UK) for peptide assignment. The criteria were in accordance with the manufacturer’s instruc- tions. If necessary, match acceptance of automated batch processes was conﬁrmed by manual inspection of each set of raw MS/MS spectra in which the major product ions were matched with theoretically predicted product ions from the database-matched peptides. A mock eluate, which used anti-FLAG with the FLAG peptide, was analyzed by the same LC-MS/MS method as used for the NOP132-associated complexes and the identiﬁed proteins were then subtracted from the proteins identiﬁed in the FLAG-NOP132 co- precipitations. Thus, those proteins identiﬁed in the mock eluate were not included in the list of NOP132-associated proteins unless a quantitative increase was conﬁrmed. Purification of GST-fusion proteins An Escherichia coli BL21 strain carrying a GST plasmid was grown in 750 ml of Luria–Bertani medium and was treated with isopropyl b-D-thiogalactoside (ﬁnal conc., 0.2 mM) for 4 h at 30 C as previously described (8). Cells were dispersed in lysis solution (1· PBS, 2 mM EDTA, 0.1% b-mercaptoethanol, 0.2 mM phenylmethyl sulfonyl ﬂuoride, and 10 mg/ml aprotinin) at a ratio of 1:5 (cell volume: lysis solution) and were sonicated on ice three times for 5 min each, with a microtip, 40% cycle, and output control of 4. After two rounds of centrifugation at 10 000 g for 10 min at 4 C, 10 ml of the supernatant was mixed with 1 ml of a 50% (v/v) slurry of glutathione-Sepharose-4B beads (GE-Healthcare) and rotated for 30 min at 4 C. The beads were washed four times with the lysis buffer. In vitro binding assay For the in vitro binding assay, a Sf9 cell lysate expressing baculovirus-produced NOP132 was obtained by infecting