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The FASEB Journal • Research Communication

Phospholipase C␤1 is linked to RNA interference of specific genes through translin-associated factor X Finly Philip, Yuanjian Guo, Omoz Aisiku,1 and Suzanne Scarlata2 Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York, USA ABSTRACT Phospholipase C␤1 (PLC␤1) is a G-protein-regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have previously found that in solution PLC␤1 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 PLC␤1 and TRAX interact in SK-N-SH cells and also in HEK293 cells induced to overexpress PLC␤1. In HEK293 cells, TRAX overexpression ablates Ca2ⴙ signals generated by G protein-PLC␤1 activation. TRAX plays a key role in down-regulation of proteins by small, interfering RNA, and PLC␤1 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 PLC␤1 in HEK293 and HeLa cells allows for an ⬃20% increase in siRNA(GAPDH) silencing. While PLC␤1 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). PLC␤1 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 PLC␤1 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 PLC␤1 in gene regulation through TRAX association.—Philip, F., Guo, Y., Aisiku, O., Scarlata, S. Phospholipase C␤1 is linked to RNA interference of specific genes through translinassociated factor X. FASEB J. 26, 000 – 000 (2012). www.fasebj.org

Key Words: G protein signaling 䡠 RNA transcription 䡠 histone H2B The G␣q family of G proteins, which are activated through many hormones and neurotransmitters to

Abbreviations: Ago2, Argonaute-2; Dcr2, Dicer 2; eCFP, enhanced cyan fluorescent protein; eYFP, enhanced yellow fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HEK, human embryonic kidney; LDH, lactate dehydrogenase; NLS, nuclear localization signal; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PLC␤, phospholipase C␤; RISC, RNA-induced silencing complex; TRAX, translin-associated factor X 0892-6638/12/0026-0001 © FASEB

transmembrane receptors, generates cell signals primarily through the activation of phospholipase C␤ (PLC␤) enzymes (for reviews see refs. 1, 2). These enzymes catalyze the hydrolysis of the signaling lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, which stimulate the release of Ca2⫹ from intracellular stores and activate protein kinase C, respectively, and lead to mitogenic and proliferative changes in the cell. Of the PLC␤ subtypes, four are known to differ in their sensitivity to G proteins and their tissue distribution. PLC␤1 is highly expressed in neuronal tissue and is the most sensitive to G␣q. PLC␤1 resides predominantly on the plasma membrane, where it is associated with G␣q (3). In addition, PLC␤1 can be found in the cytosol and the nucleus. The nuclear localization of PLC␤1 depends on the state of cellular differentiation, cell cycle, and environmental conditions (see refs. 4, 5), which may be triggered by the exposure of PLC␤1’s nuclear localization signal (NLS) and/or phosphorylation (6). The regulation and function of PLC␤1 in these alternate cellular compartments is unknown. We have found recently that the protein translinassociated factor X (TRAX) is a strong binding partner of PLC␤1 (7). Interaction between the two proteins occurs through the helical C-terminal domain of PLC␤1, which is also the binding region of G␣q, and excess TRAX can displace G␣q from PLC␤1, resulting in deactivation of the enzyme. TRAX forms strong complexes with translin (8). Translin is a singlestranded DNA- and RNA-binding protein (9) with proposed functions in chromosomal translocations in lymphoid cells, and mRNA transport and storage in brain and testis (10). TRAX binds translin in the cytosol, which allows the complex to partition into the nucleus through TRAX’s NLS (11). Recently, it was found that TRAX and translin form an octamer that activates RNA-induced silencing complexes (RISCs; 1 Current address: Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. 2 Correspondence: Department of Physiology and Biophysics, Stony Brook University, Basic Science Tower, T6-146, Stony Brook, NY 11794-8661, USA. E-mail: suzanne.scarlata@ stonybrook.edu doi: 10.1096/fj.12-213934 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

1

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refs. 12–14). In humans, siRNA down-regulation occurs through a pathway in which the duplex siRNA is guided onto the protein Argonaute-2 (Ago2) of RISC (see ref. 15). C3PO, a complex consisting of 2 TRAX:6 translin molecules, promotes RISC activity by facilitating siRNA unwinding and removal of the cleaved passenger strand. In humans, siRNA activity can be reconstituted by the TRAX-translin octamer and Ago2 (12), and unlike Drosphilia, Dicer-2 (Dcr2) is not required (12). While PLC␤1 and TRAX have different cellular functions, knockout mice of these proteins show similar neurological symptoms (e.g., refs. 16 –18). In addition, knocking out TRAX has been reported to down-regulate gene expression of several G protein regulated genes, including two G␣q-coupled receptors (17, 19). We have shown here that PLC␤1 associates to TRAX in cells, affecting its RISC activity. This effect is seen by the reversal of siRNA down-regulation by overexpressed PLC␤1 of genes to two metabolic enzymes, lactate dehydrogenase (LDH) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), that promote the transcription of histone H2B (20, 21) allowing cells to progress through the S phase. Our studies suggest a novel link between G␣q, and transcriptional regulation and cell cycle progression through PLC␤-TRAX interactions.

Western blotting The cell lysates were subjected to gel electophoresis, followed by the transfer of the samples to a nitrocellulose membrane. The membrane was then incubated for 1 h with 1% BSA to block the nonspecific binding. The primary antibody, diluted according to the manufacturer’s instructions in Tris-buffered saline with 0.5% Tween (TBST), was added to the membrane and incubated for 1 h. This was followed by 3 washes of 15 min each in TBST. After the addition of horseradish peroxidase-conjugated secondary antibody for 1 h, the membrane was washed 3 times for 15 min each in TBST. The membrane was either exposed to a photographic film or subjected to imaging after a chemiluminescence reaction (Pierce, Inc., Rockford, IL, USA). Pulldown studies HEK293-␤1 cells, treated with tetracycline for 24 h to express PLC␤1 with a streptavidin peptide tag, were washed twice with ice-cold PBS and lysed with 150 mM NaCl, 20 mM HEPES (pH 7.4), 2 mM MgCl2, 5 mM ␤-mercaptoethanol, 1 mM PMSF, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin by a homogenizer. The lysate was added to streptavidin beads and rotated for 1 h at 4°C. The beads were washed with the lysis buffer and eluted with buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM KCl, 2 mM EDTA, 5 mM ␤-mercaptoethanol, 100 ␮g/ml BSA, and 2 mM biotin. Coimmunoprecipitation

MATERIALS AND METHODS

Materials Human embryonic kidney (HEK)293-␤1 and HEK293-TAPPLC␤1 cells and enhanced yellow fluorescent protein (eYFP)PLC␤1 were generous gifts from Dr. Loren Runnels (University of Medicine and Dentistry of New Jersey, Piscataway, NJ, AQ: 1 USA). These cells were prepared by introducing PLC␤1 tagged with a 38-aa strepavidin-binding peptide on the N AQ: 2 terminus (Sigma, St. Louis, MO, USA) using the Flp-In system (Invitrogen, Carlsbad, CA, USA). It has been shown that the fluorescent tagged PLC␤1 shows wild-type behavior in transfected cells. Mouse monoclonal and rabbit polyclonal PLC␤1 antibodies and mouse monoclonal and goat polyclonal primary antibodies for TRAX were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). An additional mouse monoclonal antibody for TRAX was from BD Biosciences (San Jose, CA, USA). Primary antibodies for Dcr2 (rabbit) and Ago-2 (mouse) were from Abcam, Inc. (Cambridge, MA, USA). Cell culture HEK-␤1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum at 37°C with 5% CO2. eYFP-PLC␤1, Trax– enhanced cyan fluorescent protein (eCFP), or G␣q-RC-eCFP were transfected into HEK293 cells with FuGene-HD (Promega, Madison, WI, USA) using the manufacturer’s protocol or by electroporation using a protocol adapted from Maniatis and colleagues (22) as described previously (3). Proteins were viewed by immunofluorescence using the methodology described in ref. 23. 2

Vol. 26

December 2012

To confirm the results on TRAX pulldown by PLC␤1 through the streptavidin-based system, we performed a coimmunoprecipitation experiment using PLC␤1 antibodies on SK-N-SH cells. The cells were lysed by passing the cells 10 times through a 25-gauge needle, followed by 20 strokes of homogenization in lysis buffer containing 50 mM NaCl, 20 mM HEPES, 2 mM MgCl2, 5 mM ␤-mercaptoethanol, and protease inhibitors. The cell lysates were incubated overnight at 4°C with PLC␤1 antibodies that were prebound to proteinASepharose beads. After washing the beads 3 times in lysis buffer, the proteins were eluted from the beads in electrophoresis sample buffer at 95°C for 5 min. Separation of nuclear and cytosolic cell fractions HEK293 cells were harvested by centrifugation at 600 g for 5 min. Cells were resuspended in 5 vol of PBS at 4°C and centrifuged 5 min at 600 g. The washed pellet was resuspended in 10 vol of a hypotonic buffer solution (Active Motif Inc., Carlsbad, CA, USA) and placed on ice for 10 min. Cells were checked for swelling under a phase-contrast microscope. Once 90% had swollen, they were pipetted into a prechilled Dounce-type homogenizer and subjected to 10 –12 upward strokes. This process was continued until ⬍10% whole cells remained. The suspension was centrifuged at 1000 g for 3 min to pellet the nuclei, and the pellet was resuspended in 10 vol of the hypotonic buffer solution. This latter step was repeated twice. Verification of the nuclear and cytosolic extraction was additionally performed using a commercial kit from Thermo Scientific (Waltham, MA, USA) and following the manufacturer’s protocol. siRNA studies siRNA for GAPDH, hsp90, cyclophilin A, translin, PLC␤1, and negative control were purchased from Dharmacon Inc.

The FASEB Journal 䡠 www.fasebj.org

PHILIP ET AL.

tapraid4/z38-faseb/z38-faseb/z3801212/z388989d12z xppws S⫽1 8/7/12 1:07 Ms. No.: 12-213934 Input-ACT (Lafayette, CO, USA) The purchased siRNAs are from the company’s On-Target Plus products, consisting of a mixture of four different RNA sequences that target different regions of the specific mRNA with a low probability of off-target interactions. Proteins were down-regulated following the manufacturer’s protocol. PLC␤1expression in HEK293-␤1 was induced by 1 ␮g/ml tetracycline for 40 –50% confluence 24 h before knockdown. Untreated cells were used as control. Live cells were counted at 12, 24, 48, and 72 h postknockdown and were lysed after harvesting. Electrophoresis was done on both detached cells and attached cell lysate to check for protein expression. Caⴙ2 measurements Tetracycline-treated HEK293-␤1 cells that were transfected with eCFP-G␣q and/or TRAX-eCFP (to assess transfection efficiency) and harvested after 48 h. Cellular calcium levels were determined using Fura-2-AM (Invitrogen) using an ISS spectrofluorometer (ISS Inc., Champaign, IL, USA) as described previously (24). RT- PCR RNA was extracted by the RNeasy mini system (Qiagen, Valencia, CA, USA) followed by cDNA synthesis with the use of QuantiTect Reverse Transcriptase (Qiagen). The primers were from Applied Biosciences (Foster City, CA, USA). Quantitative real-time PCR was performed on the cDNA samples using TaqMan method (Applied Biosciences) on an MJ Research Opticon II system (MJ Research. St. Bruno, QC, Canada). The GAPDH expression levels in each sample were normalized to ␤-actin or ubiquitin levels. The relative expressions of GAPDH mRNA levels were calculated using the comparative CT method (2⫺⌬⌬Ct; see ref. 25). RNA gel electrophoresis Electrophoretic mobility of RNA samples of same concentration was monitored on a 2% agarose gel. Ethidium bromide fluorescence intensities of bands lower than 600 bp were quantified for both groups using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA) and compared using a paired t test.

RESULTS PLC␤1 and TRAX are associated in HEK293 cells

F1

We have found previously that purified PLC␤1 and TRAX strongly associate in solution, and endogenous as well as overexpressed PLC␤1 and TRAX are colocalized in the cytosol and nucleus of C6 glial cells and Neuro2A cells (7). Here, we used wild-type HEK293 (wtHEK29) cells or ones containing a PLC␤1 gene whose expression is under regulation of a tetracycline promoter (HEK293-␤1; see Materials and Methods). Figure 1A shows the colocalization of eYFP-PLC␤1 in transfected wtHEK293cells and endogenous TRAX, visualized using a polyclonal antibody. In accord with previous studies (3), PLC␤1 localizes primarily to the plasma membrane but still has a significant cytosolic population. Alternately, TRAX is found in the cytosol and nucleus but not the plasma membrane. The abPHOSPHOLIPASE C␤ IS LINKED TO RNA SILENCING

sence of TRAX on the plasma membrane is thought to be due to a lack of intrinsic membrane affinity, as well as competition for PLC␤1 binding sites by G␣q (7). Cellular association of PLC␤1 and TRAX was verified by probing for TRAX after immobilizing PLC␤1, which has a streptavidin peptide tag that can bind to streptavidin beads, expressed in tetracycline-treated HEK293-␤1 cells (Fig. 1B). To support the idea that endogenous PLC␤1 and TRAX associate in cells, we carried out coimmunoprecipitation studies in SK-N-SH cells that express easily detectable levels of these proteins. The results in Fig. 1C suggest association between the proteins. In another series of studies, we used Förster resonance energy transfer to verify the association of fluorescent-tagged PLC␤1 and TRAX expressed in HEK293 cells; these results are included in Supplemental Data. In vitro studies showed that TRAX competes with G␣q for PLC␤1 binding and attenuates its activation (7). To determine whether this competition occurs in cells, we transfected tetracycline-treated HEK293-␤1 cells with TRAX and cotransfected with G␣q to provide enough G protein for the overexpressed PLC␤1. We found that TRAX eliminates Ca2⫹ signals produced by carbachol stimulation (Fig. 1D) supporting the idea that TRAX binds to PLC␤1 intracellularly to reverse G␣q activation. G␣q stimulation promotes movement of a population of TRAX to the nucleus In addition to its role in RNA silencing in the cytosol, TRAX has proposed roles in functions in the nucleus (10). Since G␣q and TRAX bind to the same region of PLC␤1, and since the affinity between PLC␤1 and G␣q increases ⬃20-fold with activation, activating G␣q should disrupt TRAX-PLC␤1 complexes, which can result in changes in TRAX localization. This idea was tested by treating HEK293-␤1 cells with tetracycline to induce PLC␤1 expression, stimulating with carbachol for 5 min or 30 min (both gave identical results), and following localization by cell fractionation and Western blotting. While we could not detect movement of PLC␤1, we found that a population of TRAX moved to the nucleus (Fig. 2). This result correlates well with the F2 observation that carbachol stimulation results in a 46% drop in TRAX-PLC␤1 colocalization that may accompany movement of TRAX with stimulation (Table 1), T1 and also immunofluorescence studies support changes in TRAX properties with G␣q activation (Supplemental Data). These studies suggest that TRAX localization is responsive to G protein activation. TRAX has been found to bind to the long cytosolic tail of adenosine 2A receptors. These receptors are coupled to G␣i/o subunits. We tested whether activation of these receptors would similarly result in changes in TRAX localization. Stimulation of HEK293-␤1 untreated or treated with tetracycline with adenosine did not affect the localization of TRAX (Supplemental Data). In addition, TRAX does not appear to affect the cellular localization of PLC␤1 in the basal and stimulated states of tetracycline-treated HEK293-␤1 cells, suggesting that TRAX interaction does not significantly 3

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A

B

Colocalization between eYFP-PLCβ1 and endogenous TRAX in HEK293 cells

Pull-down of PLCβ1 by streptavidin beads in HEK293-β1cells

PLCβ1 No Tet

Tet

No Tet

Flow-through Anti-Trax-Alexa-647

eYFP-PLCβ1

C

Merge

PLCβ1 levels in eluates and lysates in Co-IP experiment

TRAX levels in Co-IP experiment with PLCβ1 20000

p=