Fig 2: NOPCHAP1 enhances expression of NOP58. (A) Characterization of a NOPCHAP1 KO clone generated in human HEK 293T cells. Western Blot with anti-NOPCHAP1 antibody using Laemmli extracts from NOPCHAP1 KO clone (KO) or control clone (Ct). Anti-GAPDH antibody was used as loading control. (B) The nucleolar structure is not affected in the NOPCHAP1 KO clone. Epifluorescence microscopy images of HEK 293T control and NOPCHAP1 KO clones. Red/left panels: RNA FISH with U3 specific probes. Green/ middle panels: IF with an anti-NOP58 antibody. Right panels: merge with DAPI staining. Scale bar is 10 µm. (C) Top panel: schematic representation of the assay. Bottom panel: graph showing the ratio FL/RL representing the expression of indicated FL-fusion protein in control cells (dark blue) and NOPCHAP1 KO cells (light blue), in normal conditions or after Geldanamycin treatment (GA) for 12 hr. The WT or mutant forms of protein used in the assay is indicated below the graph. A co-transfected plasmid expressing RL alone was used to normalize the value obtained for FL-fusion proteins. The values are means of four experiments that are each represented by a dot. Error bars: standard deviation. * P-value = 0.05 according to a Student test. (D) Model for the assembly of PRPF31 and NOP58 and specific role of NOPCHAP1 in box C/D snoRNP biogenesis. Newly-synthesized NOP58 associates with HSP70 and HSP90, and is transferred to R2TP by NOPCHAP1. A SNU13/ZNHIT3/NUFIP1 module binds R2TP via PIH1D1, and this leads to the assembly of NOP58 with SNU13, maintained by ATP-loaded RUVBL1/2, ZNHIT6 and NUFIP1/ZNHIT3. In the case of PRPF31, it is unclear how this protein associates with R2TP. The steps where NOP58 and PRPF31 mutants accumulate are indicated.

Fig 3: SPR binding assays between NOPCHAP1 and RUVBL1/2. The graphs depict the immobilized NOPCHAP1 response upon injection of RUVBL1/2 in the presence and absence of 100 μM ATP-γ-S (A and C, respectively) and RUVBL1-ΔDII/RUVBL2-ΔDII (B) (t = 0 s) at different concentrations or upon washing (t = 220 s). X-axis: time (s); Y-axis: response (arbitrary units). All assays were performed in triplicates.

Fig 4: 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 5: NOPCHAP1 bridges NOP58 CC-NOP domain to RUVBL1. (A) Left panel: schematic representation of the LUMIER-IP assay. Right panel: graph plotting the % of co-IP efficiency for the interaction between 3xFLAG-FL-NOP58 and RL-RUVBL1 using NOPCHAP1 as a third co-expressed fusion or PHAX (unrelated protein for a negative control). The values are means of four experiments that are all 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) Schematic representation of human NOP58 protein sequence. CC and NOP domains are represented. Below are the truncation mutants used for the LUMIER IPs shown in C. (C) Left panel: schematic representation of the LUMIER-IP assay. Right panel: graph plotting the % of co-IP efficiency for the interaction between 3xFLAG-FL-NOP58 fragments and RL-RUVBL1 using NOPCHAP1 as a third co-expressed fusion or PHAX as a negative control. The 3xFLAG-FL-NOP58 fragments used in the assay are indicated below the graph. Legend as in A. (D) Left panel: schematic representation of the LUMIER-IP assay. Right panel: graph plotting the % of co-IP efficiency for the interaction between RL-NOP58 and 3xFLAG-FL-RUVBL1 or RUVBL2 (WT or mutants, as indicated below the graph). Legend as in A.