Figure 3: The ATPase activity of hCINAP is orchestrated by RPS14 and essential for Nob1-mediated 18S rRNA processing.

(a) hCINAP-depleted cells were transfected with wild-type or ATPase-defective hCINAP, after which 40S subunit formation was detected by sucrose density gradient centrifugation. (b) Structural superposition of hCINAP-D77G (yellow) with hCINAP-ADP (light blue, PDB code: 5JZV). The 2F_o–F_c map of ADP in the structure of hCINAP-D77G-ADP is contoured at 1.4 ó (shown in the box). The side chain of D77 and a phosphate group in hCINAP-ADP are also shown. (c) 293T cells were transfected with Flag-hCINAP and the mutants or truncation of HA-RPS14. Cells were harvested and the interaction between hCINAP with RPS14 was examined with indicated antibodies. (d) Pull-down assay was carried out to detect the effect of hCINAP on the binding capacity of RPS14 with 18S-E pre-rRNA. (e) Thin-layer chromatography analysis was performed with γ-[³²P]ATP to examine the effect of RPS14 on the ATPase activity of hCINAP. Radioactively labelled products were visualized using a phosphor screen. (f) In vitro pull-down assay was performed to detect the interaction between hCINAP with RPS14 in the presence of ATP or non-hydrolysable ATP homologue AMP-PNP. (g) Determination of hCINAP and Nob1 interaction by GST pull-down experiments. (h) In vitro RNA cleavage assay to detect 18S-E pre-rRNA cleavage by Nob1 with wild-type hCINAP or the enzymatic mutants of hCINAP. (i) A model of regulation of 18S rRNA processing by hCINAP. In the pre-40S particles, hCINAP binds to the C-terminal of RPS14 and blocks binding of RPS14 to premature 18S rRNA, which may lead to a conformational change in the pre-rRNA. Meanwhile, RPS14 stimulates the ATPase activity of hCINAP, whereas ATP hydrolysis by hCINAP dissociates the hCINAP–RPS14 complex, which may facilitate proper assembly of RPS14 with rearranged pre-rRNA. Subsequently, hCINAP interacts with Nob1 and activates Nob1-mediated 18S rRNA processing.