Hydrolysis Rates of Different Small Interfering RNAs ( siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ

Background: PLC reverses siRNA down-regulation possibly by binding to the RNA silencing promoter C3PO. Results: PLC binds externally to C3PO to reduce rapid hydrolysis of specific siRNAs. Conclusion: A model for PLC effects on C3PO-mediated siRNA hydrolysis is presented. Significance: The studies provide a clue of the genes that may be regulated by cytosolic PLC. C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase C (PLC), which transmits G protein signals from many hormones and sensory inputs. The association between PLC and TRAX is thought to underlie the ability of PLC to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLC, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLC binds approximate to 5-fold more weakly to translin than to TRAX but approximate to 2-fold more strongly to C3PO. PLC does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLC binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLC binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLC is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLC toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.