Nucleic Acids Research, 2006, Vol. 34, No. 16 4599 Table 2. Putative NOP132-, DDX47-, and DDX18-associated trans-acting factors involved in ribosome biogenesis Putative function 18S rRNA (40S subunit) 28S rRNA (60S subunit) NOP132/NOL8-associating proteins DDX47 (RRP3), FBL(NOP1), NCL(NSR1) DDX47, GRWD1(RRB1), NPM1, WDR57 (RSA4) Other NOP254/C21orf108 DDX47-asssociating proteins BMS1L(BMS1), C4orf9(NOP14), C20orf6(ESF1), CIRH1A(UTP4), DDX47, FBL, FLJ12949 (KRI1), HEATR1 (UTP10), HRB2(KRR1), MPHOSPH10 (MPP10), NCL, NOP5 (NOP5), PDCD11 (RRP5), SART3(NSR1), TBL3(UTP13), WDR46(UTP7) BOP1(ERB1), BXDC2 (BRX1), DDX24 (MAK5), DDX27(DRS1), DDX47, EBNA1BP2(EBP2), FTSJ3(SPB1), GNL2 (NOG2), GNL3 (NUG1), GTPBP4(NOG1), GRWD1, KIAA0179 (RRP1), MKI67IP(NOP15), NOC2L(NOC2), NOC3L(NOC3), NOL1(NOP2), NOL5A (SIK1), NPM1, PES1 (NOP7), PPAN (SSF1), RBM28 (NOP4), RRS1(RRS1), RSL1D1(CIC1), XRN2(RAT1) DDX10 (HCA4), KIAA0690(RRP12), NOP254/ C21orf108, SDAD1(SDA1) DDX18-associating proteins DDX18 (HAS1), DDX47, FBL, NCL, FLJ12949 GNL3, GTPBP4, NOL1, NPM1, RSL1D1 Gene names according to HUGO nomenclature. DDX47- and DDX18-associated proteins that were also identified in FLAG-NOP132-associated complexes are underlined. DDX18-associated proteins that were also identified in FLAG-DDX47-associated complexes are in bold letter. Potential Yeast homologs are indicated in parentheses. complex but, we did not detect DDX18 in the DDX47 complex (Figure 1b, Table 1 and Supplementary Table 1), suggesting that a major portion of the DDX47 complexes is present in human cells without DDX18 or expression level of DDX18 is low. The analyses further indicated that 32 non-ribosomal proteins, including probable trans-acting fac- tors involved in ribosome biogenesis, such as nucleolin/ NCL (Nsr1), B23/NPM1, putative nucleotide binding protein estradiol-induced nuclear GTPase/GNL3 (Nug1), RNA helicase Gu/DDX21, and DHX9, found in the NOP132- associated complex were also found in DDX18- and DDX47-associated complexes (Table 1 and Supplementary Table 1). In addition most of the ribosomal proteins found in the NOP132-associated complexes were also present in the in DDX18- and DDX47-associated complexes. The overlap of proteins present in all three complexes suggests functional similarity among these complexes in human ribosome biogenesis. Among the non-ribosomal proteins we identiﬁed, karyo- pherins a3, a4, and b1 were present only in NOP132- associated complexes (Table 1 and Supplementary Table 1). Of these, karyopherin a3 interacted with NOP132 even after- RNase treatment (data not shown and Supplementary Table 1). Because karyopherin a3 binds to the NLS and is a transporter for nuclear proteins containing a NLS (33), it might have a role in the transport of NOP132 from the cyto- plasm to the nucleus. Similarly, karyopherin a2 was present in the DDX47-associated complex (Figure 1b, Table 1), sug- gesting that it might be a nuclear transport factor for DDX47. Association of NOP132 with DDX47 and DDX18 Despite the fact that DDX18 and DDX47 were detected in FLAG-NOP132-associated complexes by nano-LC-MS/MS analysis, NOP132 was not found in either FLAG-DDX18- or FLAG-DDX47-associated complexes (Figure 1b and c; Table 1). To conﬁrm the association of NOP132 with DDX18 and DDX47, we performed co-immunoprecipita- tion experiments using cell extracts prepared from 293 cells transfected with NOP132 and either FLAG-tagged DDX18 or FLAG-tagged DDX47 (Figure 2a). These experiments conﬁrmed that NOP132 can associate with both DDX18 and DDX47. The association of NOP132 with DDX47 and DDX18 was decreased signiﬁcantly by RNase treatment (Figure 2a, lanes 7 and 9), suggesting that their interaction is dependent on RNA. Similarly, most of the NOP132-, DDX47-, and DDX18-associated proteins were released by RNase treatment (Figure 2b, lanes 2, 4 and 6), suggesting that RNAs are crucial for complex formation. However, several proteins remained associated with FLAG-NOP132 or FLAG-DDX47 after RNase treatment, as shown in Figure 2b (lanes 2 and 4).Other protein bands in Figure 2b (lanes 2, 4 and 6) were identiﬁed to be NOP132, DDX47, DDX18 or background proteins (identiﬁed from a control experiment of 293 cells transfected with a FLAG vector) (Figure 2b, lanes 8 and 9). We also examined the associa- tion using puriﬁed GST-DDX47, GST-DDX18 and GST- GRWD1 with NOP132 synthesized in baculovirus in an in vitro binding assay and demonstrated that NOP132 bound both DDX47 and DDX18 in vitro (Figure 2c). A very small but signiﬁcant amount of NOP132 was detected when a small amount of GST-DDX18 was used to pull down NOP132. We were unable to use larger amounts of GST-DDX18 because most of the E.coli-produced GST- DDX18 was insoluble. Although GRWD1 was detected in NOP132-associated complexes that were isolated from cell lysates, GST-GRWD1 was not able to bind NOP132 in vitro (Figure 2c, lane 2), suggesting that the association of GRWD1 with NOP132 occurs indirectly. Alternatively, it could require a co-factor that was not present in this in vitro system. Control GST did not pull down NOP132 (Figure 2c, lane 1). Because NOP132 bound FLAG-DDX47 in vitro, the failure to identify NOP132 in the DDX47- associated complexes by mass spectrometry suggests that their association is transient or that NOP132 is only a minor component of DDX47-associated complexes. Next, we examined whether NOP132 co-localized with DDX47, DDX18, RPL3, or KIAA0539/NOP254/C21orf108