Phospholipase Cβ1 is linked to RNA interference of specific genes through translin‐associated factor X

Abstract

Phospholipase Cβ1 (PLCβ1) is a G-protein-regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have previously found that in solution PLCβ1 binds to the RNA processing protein translin-associated factor X (TRAX) with nanomolar affinity and that this binding competes with G proteins. Here, we show that endogenous PLCβ1 and TRAX interact in SK-N-SH cells and also in HEK293 cells induced to overexpress PLCβ1. In HEK293 cells, TRAX overexpression ablates Ca(2+) signals generated by G protein-PLCβ1 activation. TRAX plays a key role in down-regulation of proteins by small, interfering RNA, and PLCβ1 overexpression completely reverses the 2- to 4-fold down-regulation of GAPDH by siRNA in HEK293 and HeLa cells as seen by an ∼4-fold recovery in both the transcript and protein levels. Also, down-regulation of endogenous PLCβ1 in HEK293 and HeLa cells allows for an ∼20% increase in siRNA(GAPDH) silencing. While PLCβ1 overexpression results in a 50% reversal of cell death caused by siRNA(LDH), it does not affect cell survival or silencing of other genes (e.g., cyclophilin, Hsp90, translin). PLCβ1 overexpression in HEK293 and HeLa cells causes a 30% reduction in the total amount of small RNAs. LDH and GAPDH are part of a complex that promotes H2B synthesis that allows cells to progress through the S phase. We find that PLCβ1 reverses the cell death and completely rescues H2B levels caused by siRNA knockdown of LDH or GAPDH. Taken together, our study shows a novel role of PLCβ1 in gene regulation through TRAX association.

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

“…In an effort to understand this cytosolic population, we searched for novel protein partners and identified the protein translin-associated factor X (TRAX) through a yeast two-hybrid study. Subsequent work verified that TRAX and PLC␤ associate both in solution and in cells (8,9). We also found that TRAX competes with G␣ q for PLC␤ binding, and overexpression of TRAX ablates Ca 2ϩ signals mediated through G␣ q /PLC␤ (8).…”

“…Subsequent work verified that TRAX and PLC␤ associate both in solution and in cells (8,9). We also found that TRAX competes with G␣ q for PLC␤ binding, and overexpression of TRAX ablates Ca 2ϩ signals mediated through G␣ q /PLC␤ (8).…”

“…Our recent studies have indicated that in cells PLC␤ interferes with C3PO function, presumably through its interaction with TRAX (8). Specifically, we found that PLC␤ can reverse siRNA-mediated down-regulation of the housekeeping en-zymes, GAPDH and LDH, although overexpression of TRAX or an activated form of G␣ q reverses this effect.…”

“…We have found that PLC␤ and TRAX bind strongly in solution and form complexes in cells (8,9), and here we determined the distribution of eYFP-PLC␤⅐eCFP-TRAX complexes using FRET. We find a very high and uniform range of FRET values throughout the cytosol (FRET ϭ 24.4 Ϯ 0.3% (n ϭ 36)).…”

Emerging role for PLCβ1 MiRNA and disease

“…It has been demonstrated that PLCβ1 reduces the nuclease activity of C3PO and TRAX by an amount that depends on RNA sequence and structure. Changes in microRNAs (miRNAs) occurs with PLCβ expression and some miRNAs are significantly down-regulated when PLCβ1 is over-expressed in HEK29 cells are closely linked to various leukemias and lymphomas in that PLCβ1 expression is directly linked to these diseases [45,46] . A group of researchers identified PLCβ1 as a, expected, conserved target of miR-26b [47] .…”