Phospholipase C Beta Reduces The Binding And Cleavage Of Oligonucleotides By Component 3 Promoter Of Risc

Phospholipase C beta1 (PLCβ1), a key component in the classical pathway of G protein signaling system, relays information from G-proteins to downstream effectors upon stimulation of G-protein coupled receptors. Recently we found a novel role for PLCβ1 in gene regulation through TRAX, a protein implicated in RNA interference pathway. TRAX in complex with its protein partner translin, a DNA and RNA binding protein, has nuclease activity. We found that in cells, PLCβ1 selectively reverses the siRNA-mediated down-regulation of certain housekeeping genes such as GAPDH and LDH, but not of Cyclophilin A or Hsp90. Over-expression of TRAX inhibited the PLCβ1-mediated rescue of down-regulation of GAPDH and LDH. Our data in HEK and Hela cells indicate that PLCβ1 might play a role in TRAX-mediated RNA-induced silencing complex (RISC) activities of metabolic proteins. We have carried out in-vitro studies to characterize the biochemical properties of TRAX and translin, and the effect of PLCβ1 on binding and rate of hydrolysis of oligonucleotides by TRAX and TRAX-translin octameric complex, Component 3 promoter of RISC (C3PO). From acrylamide as well as agarose native gel electrophoresis and anisotropy measurements, we observed that translin, TRAX and C3PO exist in multiple oligomeric states that bind DNA and RNA. We were able to determine the cleavage rates for ssRNA by TRAX, translin and C3PO. We found that PLCβ1 reduces the rate of hydrolysis by C3PO through decreasing the affinity of a weak binding site of RNA on C3PO complex. The effect of PLCβ1 is seen at higher concentration of C3PO suggesting a role for PLCβ1 at localized elevated levels of C3PO, as in RISC.