PMC

Figure 3. Inorganic phosphate inhibits fly Dicer-2 from cleaving pre-miRNA. Dicer-2 cleaves pre-miRNA in the absence of inorganic phosphate, and inorganic phosphate inhibits this pre-miRNA cleavage activity. Inorganic phosphate does not affect pre-miRNA cleavage by Dicer-1 or long dsRNA cleavage by Dicer-2.

Data are presented as individual (black dots, n = 13) and group mean (grey bars) for the peak increase in mean arterial blood pressure (MAP) resulting from an injection of lactate (LA, 24mM, pH 2.66 or 6.0), inorganic phosphate (Pi, 86mM, pH 6.0) or lactate combined with inorganic phosphate (Pi/LA, pH 6.0). Sum (LA + Pi), artithmetic sum of the peak pressor responses evoked by separated injections of LA and Pi; ***, P < 0.001 compared to lactate at a pH of 2.66; †, P < 0.05 compared to Pi at a pH of 6.0; ††, P < 0.01 compared to Pi at a pH of 6.0; †††, P < 0.001 compared to Pi at a pH of 6.0; ##, P < 0.01 compared to lactate combined with Pi at a pH of 6.0.

Relationship between the amounts of phosphate intake (mg/day) from different sources (Y axis):animal (A), vegetal (B), and inorganic (C), and the total phosphate intake (mg/day) (X axis).Tertiles of phosphate intake and the tertiles of the different sources of phosphate are shown as dotted line in the Y and X axis. P: phosphate.

Figure 6. Models for the inhibition by inorganic phosphate of fly Dicer-2 processing of short, but not long, dsRNA and the role of the phosphate-binding pocket in siRNA loading. The PAZ domain of fly Dicer-2 has binding pockets for the 5′-monophosphate and the 2-nucleotide 3′-overhang of a pre-miRNA substrate. An inorganic phosphate molecule occupies the binding pocket in place of the 5′-monophosphate of the pre-miRNA, blocking its binding. Long dsRNA is recognized mainly by the helicase domain and/or the dsRBD domains and does not need a specific terminal structure to be efficiently cleaved. Long dsRNA is not blocked by the bound inorganic phosphate, since the strong interaction with the helicase domain or the dsRBD domains allows it either to bind tightly enough to Dicer-2 without additional interactions with the PAZ domain or to displace the inorganic phosphate from the binding pocket. The phosphate-binding pocket binds 5′-monophosphate of siRNA duplex in the loading step. The bound inorganic phosphate is displaced by the siRNA.