Fig 2: Characterization of NOPCHAP1. (A) Epifluorescence microscopy images of HeLa cells expressing GFP-NOPCHAP1. Blue/left panel: DAPI staining; Green/middle panel: GFP. Scale bar is 10 μm. (B) Schematic representation of SILAC IP experiments shown in C and D. (C) Proteomic analyses of the partners of GFP-NOPCHAP1. This IP is done without RNase treatment. Full hit list with Significance B values are given in Supplementary Table S3. Legend as in Figure 1 for graphic representation. (D) Comparison of GFP-NOPCHAP1 interactome in presence or in absence of RNase. SILAC ratios from condition with RNase (y axis, shown in Figure S2A) or without RNase (x axis, shown in C) were plotted against each other. (E) Scheme showing groups of NOPCHAP1 interactants found in our SILAC IPs and grouped according to the known complexes in which they belong. (F) Conservation of NOPCHAP1, NUFIP1, RPAP3-Cter (Cterminal domain) and NOP58 across Eukaryotes. Members that were not found are indicated by ‘x’. In Alveolates, NOPCHAP1 and NUFIP1 were not found in Perkinsea.

Fig 3: NOPCHAP1 interacts with WT and mutant forms of RUVBL1/2 and NOP58. (A) Left panel: schematic representation of the LUMIER-IP assay between NOPCHAP1 and RUVBL proteins. Right panel: graph plotting the % of co-IP efficiency for the interaction between co-expressed 3xFLAG-FL-NOPCHAP1 and RL-RUVBL1 or RL-RUVBL2, either WT or mutants as indicated below the graph. % of co-IP was calculated from the co-precipitated RL fusion protein (IP/Input ratios of RL activity), normalized with IP/Input ratio of FL activity obtained in the anti-FLAG IP. The values are means of four experiments, each represented by a dot. Error bars: standard deviation. Stars: values significantly greater than six-times the mean value obtained in the control IPs without anti-FLAG antibody (Control IP: light blue bars). *** P-value ≤ 0.001, ** P-value ≤ 0.01 and * P-value ≤ 0.05. (B) Left panel: schematic representation of the LUMIER-IP assay between NOPCHAP1 and NOP proteins. Right panel: graph plotting the % of co-IP efficiency for the interaction between 3xFLAG-FL-NOPCHAP1 and RL-PRPF31, RL-NOP58 or RL-NOP56 either WT or mutants as indicated below the graph. Alix is an alias name for human protein PDCD6IP and is used as a negative control. Legend as in (A). *** P-value ≤ 0.01.

Fig 4: In vitro characterization of a NOP58:NOPCHAP1:RUVBL1/2 complex. (A) Coomassie-stained SDS-PAGE 10% of His-NOP58-CC-NOP co-expressed with NOPCHAP1 in E. coli and purified on TALON resin. From left to right: M: size of the markers in kDa; SN: soluble fraction; B: bound on resin; E: imidazole eluate. *: non-specific protein (Chaperone protein DnaK) from E. coli extract. (B) Coomassie-stained SDS-PAGE 10% of His-NOPCHAP1 co-expressed with RUVBL1/2 in E. coli and purified on TALON resin. Legend as in A. FT: flow through. All labeled bands are identified by LC-MALDI. (C) Coomassie-stained SDS-PAGE 10% of His-NOP58-CC-NOP co-expressed with RUVBL1/2 in E. coli and purified on TALON resin. Legend as in A. (D) Coomassie-stained SDS-PAGE 10% of His-NOP58-CC-NOP co-expressed with NOPCHAP1 and RUVBL1/2 in E. coli and purified on TALON resin. Legend as in A. All labeled bands are identified by LC-MALDI. (E) Coomassie-stained SDS-PAGE 10% of His-yNOP58-CC-NOP (amino acids 159 to 412 from S. cerevisiae NOP58) co-expressed with NOPCHAP1 and RUVBL1/2 in E. coli and purified on TALON resin. Legend as in A. (F) Coomassie-stained SDS-PAGE 10% of NOPCHAP1 expressed in E. coli and purified on TALON resin. Legend as in A. (G) Coomassie-stained SDS-PAGE 10% of RUVBL1/2 expressed in E. coli and purified on TALON resin. Legend as in A. (H) Gel filtration profile of His-NOP58-CC-NOP+NOPCHAP1+RUVBL1/2 eluted and cleaved by 3C protease. The chromatogram shows the absorbance (y axis) as a function of the elution volume (x axis) of the purified NOP58-CC-NOP:NOPCHAP1:RUVBL1/2 complex on an Superose® 6 10/300 analytical column. The fractions of the majority peak between the two dashed lines were analyzed by SDS-PAGE. (I) Coomassie-stained SDS-PAGE 10% of the various gel filtration fractions from H. The gel shows the major complex eluted from the column. From left to right: I: injection; M: size of the markers in kDa; Fraction numbers from 10 to 17. *: non-specific protein (Chaperone protein DnaK) from E. coli extract.

Fig 5: Identification of early assembly intermediates with NOP58 mutant proteins. (A) Epifluorescence microscopy images of HeLa cells expressing wild type (WT) or mutant GFP fusions of NOP58 (NOP58-KA/AR or NOP58-A283P). Blue/left panels: DAPI staining; Green/middle panels: GFP. Scale bar is 10 μm. (B) Schematic representation of SILAC IP experiments shown in C, D and E. First experiment was done with three conditions (L1: light label for control done with parental HeLa cells; M1: medium label for GFP-NOP58-WT; H1: heavy label for GFP-NOP58-A283P) and second with two conditions (L2: light label for control; M2: medium label for GFP-NOP58-KA/AR). (C–E) Proteomic analyses of the partners of GFP-NOP58-WT (C), GFP-NOP58- A283P (D) and GFP-NOP58-KA/AR (E). Graphs display SILAC ratios (y axis, specific versus control IP) as a function of signal abundance (x axis, log10(intensity)). Each dot represents a protein and is color encoded according to the classification shown between panels C and D. The labeled dots highlight proteins relevant to this study. Full hit list with Significance B values are given in Supplementary Table S1. (F) Bar plot comparing SILAC ratios shown in C, D and E for a few selected partners. In each experiment, the normalization is done by dividing the SILAC ratio of the protein of interest with that of NOP58. Blue bars are for NOP58-A283P, red bars for NOP58-KA/AR and green bars for NOP58-WT.