Regulation of Luteinizing Hormone Receptor Expression

Our previous studies have identified a luteinizing hormone receptor (LHR) mRNA-binding protein (LRBP) that binds to the coding region (LBS) of rat LHR mRNA. The identity of LRBP was later established as mevalonate kinase (MVK). The present study examined if LRBP binding to LHR mRNA impairs translation. A full-length FLAG-tagged rat LHR mRNA was synthesized and translated in vitro. The translation product was immunoprecipitated and analyzed on SDS-PAGE. The addition of LRBP inhibited LHR mRNA translation. This inhibitory effect was reversed by an excess of wild type (wt) LBS. To determine whether this reversal of the inhibitory effect of LRBP was indeed due to the sequestration of LRBP by the wtLBS, a translation reaction was performed in the presence of mutated LBS in which all the cytidine in the wtLBS was mutated to uridine. This mutation of LBS has been shown to render it incapable of interacting with LRBP. Unlike wtLBS, the mutated LBS was unable to reverse the inhibitory effect of LRBP on LHR mRNA translation. The addition of mevalonate, which has been shown to compete for LHR mRNA binding to LRBP, also reduced the extent of translation inhibition by LRBP. Endogenous association of LHR mRNA with MVK was assessed by immunoprecipitation of the ribonucleoprotein complex with MVK antibody followed by reverse transcription-PCR of the RNA associated with the immune complex. Amplification of LHR mRNA, if any, associated with the immunoprecipitate obtained from ovarian ribonucleoprotein complex with gene-specific primers confirmed the association of LHR mRNA with MVK. Collectively, the present data support the novel function of LRBP as a translational inhibitor of LHR mRNA in the ovary.

Biological actions of luteinizing hormone (LH), 2 a glycoprotein hormone crucial in regulating gonadal functions in mammals, are mediated by its interaction with specific cell surface receptors expressed primarily on the cell membranes of reproductive organs such as testis and ovary (1). Luteinizing hormone receptor (LHR) belongs to the rhodopsin/␤ 2adrenergic receptor subfamily A of G-protein-coupled receptors (2,3). In the ovary interaction of LH with LHR leads to increased production of cAMP, which in turn stimulates steroidogenesis and other specialized functions of the ovary such as ovulation (1,4).
The expression of LHR on the cell surface varies during different stages of the ovarian cycle, thereby regulating the actions of LH. In rat ovarian granulosa and luteal cells, the expression of LHR is greatly decreased by the endogenous preovulatory LH surge or by the administration of a pharmacological dose of human chorionic gonadotropin (hCG), a placental counterpart of LH (5)(6)(7)(8)(9). Previous studies from our laboratory have shown that the decline in cell surface expression of ovarian LHR seen after hCG administration is paralleled by a specific, transient loss of all four LHR mRNA transcripts (10). We have shown that this transient loss of LHR mRNA does not result from decreased transcription but occurs post-transcriptionally with a 3-fold decrease in mRNA half-life (8).
Further studies have shown that a specific LH receptor mRNA-binding protein (LRBP), identified in the ovarian cytosolic fraction, plays a regulatory role in the expression of LHR mRNA by binding to a polypyrimidine-rich, bi-partite sequence in the coding region of LHR mRNA (10,11). LRBP exhibits characteristics of a functional mRNA-binding protein with respect to expression in target cells, induction of its expression in the ovary when LHR mRNA levels are down-regulated, and also by demonstration of its ability to increase the degradation of LHR mRNA in an in vitro decay system (10 -14). Subsequent studies led to the purification and identification of this trans-acting factor as mevalonate kinase (MVK) (12). In vivo studies showed an increase in mevalonate kinase expression in rat corpus luteum before ligand-induced loss of LHR mRNA, consistent with its role as an endogenous regulator of LHR mRNA expression (14). Because the mRNA binding characteristics of this protein revealed specificity for sequences in the LHR mRNA coding region, the present studies were undertaken to examine if binding of LRBP to the coding region of LHR mRNA impairs translation. The results presented here show that in a cell-free in vitro translation system, LRBP binds to the coding region of LHR mRNA, and the resulting ribonucleoprotein complex prevents LHR mRNA translation. This was further supported by the fact that immunoprecipitation of ribonucleoprotein (RNP) complex from ovarian homogenate showed the association of LH receptor mRNA with mevalonate kinase in vivo during hCG-induced LH receptor down-regulation in the ovary.

MATERIALS AND METHODS
Chemicals-Pregnant mare serum gonadotropin was purchased from Calbiochem. Highly purified human chorionic gonadotropin (CR 127) was a gift from the Center for Population Research (NICHD, National Institutes of Health) through the National Hormone and pituitary Program. [␣-32 P]UTP was from PerkinElmer Life Sciences and Redivue L-[ 35 S]methionine (in vitro translation grade) was from Amersham Biosciences. mMessage mMachine T7 Ultra was a product of Ambion (Austin, TX). EDTA-free protease inhibitor mixture tablets and Quick spin columns (G-50-Sephadex) for radiolabeled RNA purification were obtained from Roche Applied Science. RNasin and Flexi rabbit reticulocyte lysate system were purchased from Promega (Madison, WI). Macro-Prep high S support column was from Bio-Rad. Centriplus YM-10, Centricon YM-10, and Microcon YM-10 were products of Millipore Corp. (Bedford, MA). Anti-FLAG M2-agarose affinity gel and DL-mevalonic acid lactone were purchased from Sigma. BCA reagent was from Pierce. Enlightning (rapid autoradiography enhancer) reagent was a product of PerkinElmer Life Sciences.
Animals and Tissues-Pseudopregnancy was induced in 21-day-old Sprague-Dawley female rats by subcutaneous injection of 50 IU of pregnant mare serum gonadotropin followed by 25 IU of hCG 56 h later. The day of hCG injection was taken as day 0. LH receptor down-regulation was induced by the injection of 50 IU of hCG on the fifth day of pseudopregnancy. Ovaries were collected 6 and 12 h after hCG injection and were processed immediately for immunoprecipitation of RNP complex and LRBP purification.
Purification of LRBP-LRBP from rat ovary was purified as described previously (12). Partial purification of LRBP was performed by homogenizing rat ovaries in buffer A (10 mM Hepes, pH 7.9, 0.5 mM MgCl 2 , 50 M EDTA, 5 mM dithiothreitol, and 10% glycerol) containing 50 mM KCl and protease inhibitor mixture at 4°C. The cytoplasmic proteins were collected by centrifuging the homogenate at 105,000 ϫ g for 90 min at 4°C. The supernatant containing the cytoplasmic proteins (S100 fraction) was collected and applied to a strong cation exchange column (Macro-Prep High S Support) equilibrated with buffer A containing 50 mM KCl. The column was washed with buffer A containing 50 mM KCl until the absorbance at 280 nm was less than 0.02. The partially purified LRBP was then eluted with buffer A containing 150 mM KCl and desalted to 50 mM KCl using Centricon YM-10 microconcentrators. The protein concentration was determined by BCA. The final purification was performed by separating the partially purified LRBP on SDS-PAGE and eluting the LRBP from the gel and renaturing as described before (12). Briefly, partially purified LRBP preparation was treated with SDS sample buffer at 37°C for 10 min and separated on a 10% SDSpolyacrylamide gel under a constant voltage of 150 V. The gel strip containing the LRBP was cut with a razor blade and placed in a 1.5-ml Eppendorf tube. The gel piece was then homogenized in elution-renaturation buffer (1% Triton X-100, 20 mM Hepes, pH 7.6, 1 mM EDTA, 100 mM NaCl, 2 mM dithiothreitol) with a Teflon pestle and incubated for 2 h at 37°C. After incubation, the sample was centrifuged at 10,000 ϫ g for 5-10 min at 4°C to sediment the residual polyacrylamide. The supernatant containing the LRBP was collected, buffer-exchanged with buffer A containing 50 mM KCl to remove TritonX-100, and concentrated using Microcon centrifugal filter device.
Templates for in Vitro Transcription-The pCMV4 vector containing 2.1-kilobase rat LHR cDNA (15) was used to generate the full-length rat LHR cDNA containing T7 promoter at the 5Ј end and FLAG tag at the 3Ј end. The oligonucleotide primers used for PCR were synthesized by Invitrogen, and their sequences were as follows: sense primer, 5Ј-G-CATGCTAATACGACTCACTATAGGGATGGGGCGGCGAGTC-CCAG-3Ј (underlined sequences represent the T7 RNA polymerase promoter); antisense primer, 5Ј-CTACTTGTCATCGTCGTCCTTG-TAGTCGTGAGTTAACGCTCTCGGTGG-3Ј (underlined sequences represent the FLAG tag). The cDNAs for generating the wild type and the mutant LRBP binding site (LBS) of LHR mRNA were chemically synthesized with T7 RNA polymerase promoter at the 5Ј ends. The pBluescript SK vector containing the human ␤-actin (GenBank TM accession number M77874) was purchased from ATCC and digested with BssHII to obtain the ␤-actin cDNA with T7 RNA polymerase promoter at the 5Ј end and T3 RNA polymerase promoter at the 3Ј end.
In Vitro Transcription-Unlabeled and ␣-32 P-labeled RNAs were in vitro transcribed from cDNA templates using Ambion in vitro transcription kits. The full-length capped and FLAG-tagged rat LHR mRNA and human ␤-actin mRNA were synthesized using mMessage mMachine T7 Ultra kit. The wild type and mutant LBSs were synthesized using the MAXIscript kit. The radiolabeled LHR mRNA was prepared using 60 Ci of [␣-32 P]UTP in the reaction mixture. After transcription, the RNAs were treated with RNase-free DNase 1 and extracted with nuclease-free water-saturated phenol/chloroform/isoamyl alcohol (50: 49:1). Unincorporated nucleotides were removed using Quick spin columns (G-50 Sephadex). The RNA was precipitated with an equal volume of isopropyl alcohol at Ϫ20°C. The precipitated RNA was washed 3 times with 75% ethanol, air-dried, and dissolved in nuclease-free water. Both radiolabeled and unlabeled RNAs were quantitated spectrophotometrically at 260 nm.
In Vitro Translation-In vitro translation reactions (25-l reaction volume) were performed using a Flexi rabbit reticulocyte lysate system as described by the manufacturer. Proteins synthesized in vitro were labeled with [ 35 S]methionine and separated by 10% SDS-PAGE (Bio-Rad mini gel) according to the method of Laemmli. The gel was fixed in 40% methanol (v/v) and 10% acetic acid (v/v) for 20 min and then incubated in Enlightning reagent for another 30 min. The gel was then dried under vacuum for 20 min at 80°C and exposed to x-ray film for autoradiography.
Immunoprecipitation-FLAG-tagged in vitro translated rat LH receptor was immunoprecipitated using anti-FLAG M2-agarose affinity gel. 25 l of the in vitro translation reaction mixture was diluted to 500 l with dilution buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100). Anti-FLAG M2-agarose affinity gel was washed 3 times with wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) and added to the diluted translation reaction mixture (40 l of gel suspension per 500 l of diluted translation reaction mixture) and incubated overnight in an end-over-end shaker at 4°C. The sample was centrifuged for 5 s at 10,600 ϫ g at room temperature, and supernatant was removed. The beads were washed 3 times with wash buffer, and 30 l of 2ϫ SDS-PAGE sample buffer was added. The beads with sample buffer were heated at 65°C for 20 min and centrifuged at 10,600 ϫ g for 5-10 s, and the supernatant was collected. The supernatant was then applied onto a 10% SDS-PAGE.
mRNA Decay Coupled to in Vitro Translation-In vitro translation of LHR mRNA was performed with 200 ng of ␣-32 P-labeled LHR mRNA (100,000 cpm/reaction) using rabbit reticulocyte lysate system as described above in the presence or absence of LRBP. The in vitro translation reaction was stopped at 0, 20, and 40 min by adding 475 l of high salt cleaning buffer (25 mM Tris-HCl, pH 7.6, 0.1% SDS, and 400 mM NaCl) (16) to 25 l of reaction mixture. Yeast tRNA (10 g) was added per reaction to allow quantitative recovery of LHR mRNA during precipitation. The remaining RNA in the reaction mixture was extracted with an equal volume of nuclease-free water-saturated phenol and chloroform (17). The RNA was precipitated by adding an equal volume of isopropanol as described before (12). The precipitated RNA was washed 3 times with 75% ethanol and dried to remove any residual ethanol. The RNA was then resuspended in 10 l of RNA loading buffer (25 mM Tris-HCl, pH 7.6, 8 M urea, 1 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol) and analyzed on a 5% polyacrylamide, 8 M urea mini gel (Bio-Rad) (16). The gel was then dried and exposed to x-ray film for autoradiography.
Immunoprecipitation of RNP Complex-Immunoprecipitation of RNP complex was performed as described by Chu et al. (18). This technique is based on the immunoprecipitation procedure developed by Lerner and Steitz (19). Rat ovaries were homogenized in NET-2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Nonidet P-40) containing RNasin (100 units/0.5 ml of buffer) and protease inhibitor. The homogenate was centrifuged at 10,000 ϫ g for 10 min at 4°C Supernatant was collected, and 1-1.5 mg of protein/ml of the extract was precleared with 300 l of protein A-agarose beads for 30 min at 4°C in an end-over-end shaker and centrifuged at the end of the incubation period to remove the protein A-agarose beads. The cleared supernatant was then incubated with antibody for mevalonate kinase (12) along with 50 g of yeast tRNA and RNasin (100 units/0.5 ml) for 1 h at 4°C. Pre-immune rabbit IgG and high density lipoprotein-binding protein (HBP) antibody were used as negative controls. Protein A-agarose beads were then added at the end of the 1-h period and incubated further for another 30 min. The protein A-agarose immune complex was collected by centrifuging at 10,000 ϫ g for 3 min at 4°C The beads were washed 5 times with 400 l of NET-2 buffer. After the final wash 400 l of NET-2 buffer was added, and the immunoprecipitate was subjected to phenolchloroform extraction to isolate the nucleic acids associated with the immune complex. The extract was then incubated with 10 units of RNase-free DNase 1 at 37°C for 15 min followed by phenol-chloroform extraction again to isolate the RNA remaining in the extract. The RNA was then precipitated in the presence of 20 g of glycogen by isopropanol at Ϫ20°C The RNA was washed 2 times with 80% ethanol and dissolved in 15 l of nuclease-free water.
RT-PCR of Precipitated RNA-Full-length rat LH receptor DNA was synthesized by RT-PCR using Invitrogen SuperScript One-Step RT-PCR with Platinum Taq (40 cycles of PCR). Five l of the immunoprecipitated RNA was used for the amplification with the oligonucleotide primers synthesized by Invitrogen. Their sequences were as follows: sense primer, 5Ј-GCATGCTAATACGACTCACTATAGGGATGG-GGCGGCGAGTCCCAG-3Ј (underlined sequences represent the T7 RNA polymerase promoter); antisense primer, 5Ј-CTAGTGAGTT-AACGCTCTCGGTGG-3Ј.
Analysis of the Amplified DNA Sequence-RT-PCR products were separated on a 0.8% agarose gel, and the DNA bands were isolated by QIAquick gel extraction kit. The purified DNA was subjected to PCR (40 cycles) using Invitrogen PCR Platinum SuperMix for amplifying the first nearly 300 nt of rat LH receptor cDNA. The sequences of primers used were as follows: sense primer, 5Ј-GCATGCTAATACGACTCAC-TATAGGGATGGGGCGGCGAGTCCCAG-3Ј (underlined sequences represent the T7 RNA polymerase promoter); antisense primer, 5Ј-CCTTTCCAGGGAATCACTC-3Ј. The amplified DNA products were isolated after separation on a 0.8% agarose gel, and the purified products were subjected to PstI nuclease digestion. The digested DNA products were analyzed by agarose gel electrophoresis.

RESULTS
In Vitro Translation of FLAG-tagged Rat LHR mRNA-A FLAG sequence was introduced at the 3Ј end of rat LH receptor cDNA, and the corresponding mRNA was transcribed for the in vitro translation and subsequent immunoprecipitation of the translated protein as described under "Materials and Methods." To examine if the FLAG-tagged rat LH receptor mRNA could be translated in vitro, varying concentrations of FLAG-tagged LH receptor mRNA were used in the rabbit reticulocyte lysate system using 15 Ci of [ 35 S]methionine in a 25-l reaction volume. The reaction was performed for 90 min at 30°C The translation product was then immunoprecipitated using anti-FLAG M2-agarose affinity gel, separated on a 10% SDS-polyacrylamide gel, and subjected to autoradiography. The autoradiogram (Fig. 1a) revealed a single prominent band at about 63 kDa, and the intensity of this band increased with increasing mRNA concentrations from 50 to 200 ng in the translation reaction. A negative control reaction, performed with no RNA added to the translation reaction, yielded no protein band. The expected size of the non-glycosylated rat LH receptor is about 62 kDa (20). Because the FLAG epitope introduced contained 8 amino acids, FIGURE 1. Translation of FLAG-tagged rat LH receptor mRNA in vitro. a, capped, FLAG-tagged (3Ј end) rat LH receptor (rLHR) mRNA was synthesized and translated in vitro by Flexi rabbit reticulocyte lysate system using 50, 100, and 200 ng of mRNA in the presence of 15 Ci of [ 35 S]methionine for 90 min. The translated LHR protein was subsequently immunoprecipitated using FLAG antibody and separated on 10% SDS-PAGE. The gel was fixed, dried, and exposed to x-ray film for autoradiography as described under "Materials and Methods." A negative control was run with no RNA added in the reaction. b, 200 ng each of capped, FLAG-tagged, and non FLAG-tagged rat LHR mRNAs were in vitro translated using 15 Ci of [ 35 S]methionine. The translated LHR protein was immunoprecipitated using FLAG antibody and separated on 10% SDS-PAGE. The gel was then fixed and incubated in Enlightning reagent for enhancing the radiation signal for 30 min as described under "Materials and Methods." The gel was dried and exposed to x-ray film. A negative control was run with no RNA added in the reaction mixture. DECEMBER 30, 2005 • VOLUME 280 • NUMBER 52 the size of the non-glycosylated LH receptor was expected to be about 63 kDa. To further confirm that the band seen on the autoradiogram was indeed LHR protein and not a nonspecific translation product, in vitro translation was performed with LHR mRNA with and without FLAG tag at the 3Ј end. LHR protein was then immunoprecipitated from the reaction mixtures, separated by SDS-PAGE, and then subjected to autoradiography. The results presented in Fig. 2b show that the LHR protein band was visible only in the translated products of FLAGtagged LHR mRNA (lane 3). No LHR protein-specific band (lane 1 and 2) was seen when translation products were immunoprecipitated from reaction mixtures either containing no RNA or non-FLAG-tagged LHR mRNA.

Translational Regulation of LH Receptor mRNA
Effect of LRBP on the Translation of LHR mRNA in Vitro-We have previously shown that LRBP specifically binds to the coding region of LH receptor mRNA (10). To examine if binding of LRBP to LH receptor mRNA in the coding region has any effect on the translation of the mRNA, in vitro translation of LH receptor mRNA was performed in the absence and presence of LRBP at different concentrations for 90 min, and the resulting translated LHR protein was immunoprecipitated, separated by SDS-PAGE, and then subjected to autoradiography. The results (Fig. 2a) show that the LRBP caused a concentration-dependent decrease in the translation of LHR mRNA. Inclusion of an irrelevant protein, bovine serum albumin, did not have any effect on the synthesis of LH receptor protein. The translation reaction of LHR mRNA was also conducted in the presence of varying concentrations of gel-purified rat LRBP. The resulting LHR protein was analyzed as described above, and the results are shown in the Fig. 2b. The results clearly showed an inhibition of translation of LHR mRNA by as low as one g of gel-purified rat LRBP and produced almost complete inhibition at the 2-g level. A time-course of in vitro translation of LHR mRNA was then performed in the absence or presence of 1 g of gel-purified rat LRBP. The results (Fig. 2c) showed translation inhibition by LRBP at all time intervals starting at 20 min of translation reaction when compared with the respective control reactions. It should be noted that there was a slight increase in the translation of LH receptor mRNA with increase in incubation time, which is due to continued translation of LH receptor mRNA with the progress in incubation time.
To examine if this inhibition of LHR mRNA translation by LRBP was specific to LHR mRNA, human ␤-actin mRNA was in vitro translated in the presence of rat LRBP, and the resulting translation products were examined by SDS-PAGE, as described above. As shown in Fig. 3, there was no change in ␤-actin protein synthesis in the presence of LRBP, thus indicating that the inhibitory effect of LRBP was specific to the translation of LHR mRNA.
Effect of LRBP on the Translation of Rat LHR mRNA in the Presence of wtLBS and mLBS-We have previously shown that LRBP binds to a bipartite, polypyrimidine-rich sequence in the coding region of rat LHR mRNA, and all the cytidine residues in this region are required for the binding of LRBP (11). This 40-nt (188 -228) region of the rat LHR mRNA was designated as the LBS.
To further examine if the translation inhibition caused by LRBP was indeed due to the binding of LRBP to the LHR mRNA, in vitro translation reactions were performed in the presence of wild type LRBP binding site (wtLBS) or mutated (all the cytidines mutated to uridines) LRBP binding site (mLBS) of rat LHR mRNA (11) in molar excess to the full-length rat LHR mRNA. The resulting translation products were then immunoprecipitated and analyzed as described above. The results (Fig. 4) clearly indicate that the LRBP caused an inhibition of LHR mRNA translation (lanes 3 and 4) when compared with translation reactions performed in the absence of LRBP (lanes 1 and 2). The inhibition in translation produced by LRBP was decreased by the inclusion of wtLBS in the reaction in a concentration-dependent manner (lanes 5 ,  6 and 7). The addition of mLBS, which does not bind to LRBP (11), did not cause any change in the inhibition of translation of LHR mRNA produced by LRBP (lanes 8 -10). These data indicate that the translation inhibition caused by LRBP was due to the binding of LRBP to the LHR mRNA.
Effect of Mevalonate on the Translation of LHR mRNA-We have recently established the identity of LRBP as MVK (12). Additionally, mevalonate, the substrate for MVK, inhibited the binding of LRBP to LH receptor mRNA (12). We, therefore, examined the effect of mevalonate on the inhibition of LHR mRNA translation produced by LRBP. To test this, translation of LHR mRNA was performed in the presence of mevalonate at 0.5 and 1.00 mM concentrations, and the translation products  1, 2, and 3 g). The translated LHR protein was immunoprecipitated and processed for developing autoradiogram. c, 1 g of capped FLAG-tagged rat LHR mRNA was in vitro translated in a 125-l reaction system containing 5 g of purified LRBP. 25-l aliquots were withdrawn at 20, 30, 40, and 60 min of reaction and were processed for immunoprecipitation of LHR protein and autoradiogram. A control reaction was performed in the absence of purified LRBP.
were examined by autoradiography. Because the reported K m for mevalonate of purified rat liver MVK is 0.271 Ϯ 0.031 mM (21), mevalonate at the 0.5-1.0 mM range was used in the translation reaction to saturate the enzyme. We have also shown earlier that the addition of mevalonate at 0.05, 0.5, and 1.00 mM levels causes a decrease in binding of LRBP to LHR mRNA in a concentration-dependent manner (12). As shown in the Fig. 5, inclusion of mevalonate in the translation reaction also caused a concentration-dependent reversal of the inhibitory effect produced by LRBP (lanes 3 and 4) compared with that produced by LRBP alone (lane 2) in the reaction mixture. Mevalonate alone did not have any effect on the translation of LHR mRNA (lane 5). This further confirmed that the translation inhibition caused by LRBP was indeed due to the binding of LRBP to LHR mRNA.
Stability of Rat LHR mRNA in Rabbit Reticulocyte Lysate System in the Presence of LRBP-To further clarify that the inhibition in translation of rat LHR mRNA seen in the presence of LRBP was not due to an increased degradation of LHR mRNA, in vitro translation reactions of 32 P-labeled LHR mRNA were performed in the absence or presence of LRBP. The translation reactions were stopped at different time intervals (0, 20, 40 min), and the LHR mRNA remaining in the reaction mixture was extracted as described under "Materials and Methods." The extracted RNA was then separated by applying onto a 5% PAGE, 8 M urea gel and dried for 20 min at 80°C, and the autoradiogram was developed. The results shown in Fig. 6 indicate that no increased degradation of LHR mRNA occurred in the presence of LRBP (L0, L20, and L40) when compared with control (C0, C20, and C40). This observation confirmed that the inhibition of LH receptor protein synthesis in vitro was due to inhibition of translation of LHR mRNA and not due to increased degradation of mRNA.
Mevalonate Kinase Binds to LH Receptor mRNA Endogenously in the Ovary-Earlier studies from our laboratory have shown that hCG-induced down-regulation of rat LH receptor mRNA is paralleled with an increase in mevalonate kinase expression in the ovary (14). To verify that mevalonate kinase endogenously remain associated with LH receptor mRNA in the ovary during LH receptor down-regulated state, hCGinduced LH receptor down-regulated ovaries were homogenized in NET-2 buffer, and the RNP complex was immunoprecipitated using rat mevalonate kinase antibody as described under "Materials and Methods." The RNA isolated from the RNP complex was analyzed for the presence of LH receptor mRNA by performing RT-PCR using LH receptor cDNA-specific primers to amplify the coding region. The results in Fig. 7a show that immunoprecipitation using MVK antibody followed by RT-PCR amplification of the total RNA isolated from the A 5-l aliquot from a 25-l reaction system was analyzed on 10% SDS-PAGE, and the gel was fixed, incubated in Enlightning reagent, dried, and exposed to x-ray film.   . LHR mRNA decay associated with in vitro translation. In vitro translation of LHR mRNA was performed with 200 ng of ␣-32 P-Labeled LHR mRNA (100,000 cpm/reaction) using rabbit reticulocyte lysate system in the presence (L) or absence (C) of LRBP as described under "Materials and Methods." The reaction was stopped at different time intervals (0, 20, and 40 min), and the remaining LHR mRNA was extracted and separated on a 5% polyacrylamide, 8 M urea gel as described under "Materials and Methods." The gel was then dried and exposed to x-ray film for the autoradiogram. DECEMBER 30, 2005 • VOLUME 280 • NUMBER 52 immune complex resulted in two DNA bands (lane 1 and 5). The band B1 was similar in size to the cDNA band obtained from a vector (pCMV4) containing the rat LH receptor cDNA by PCR amplification (positive control) (lane 3 and 6). The shorter band B2 resolved at a position between the 1.6-and 2.0-kilobase DNA markers. Preimmune rabbit IgG and an irrelevant antibody (high density lipoprotein-binding protein antibody) did not produce any DNA bands (lane 2 and lane 7). To further verify the identity of these two bands as LH receptor cDNAs, the bands B1 and B2 were eluted from the agarose gel and subjected to PCR amplification with a different set of LH receptor cDNA-specific primers for amplifying approximately the first 300 nt. As shown in Fig.  7b, both B1 and B2 produced the expected size LH receptor cDNA bands (lanes 1 and 3), similar to the band produced from the rat LH receptor cDNA positive control (lane 5). These bands were then eluted and digested with restriction enzyme PstI, which has a single site in the first 300-nt LH receptor cDNA region. The results (lanes 2 and 4) indicated that these regions were in fact cleaved by PstI and resulted in products of the expected size, similar to the bands produced from the positive control (lane 6), thus confirming that the bands B1 and B2 were in fact LH receptor cDNAs. These results, therefore, indicate that mevalonate kinase indeed forms RNP complexes with LH receptor mRNA in the ovary.

DISCUSSION
In addition to the regulation of the rate of RNA synthesis, controlling the stability of mRNA is another effective means of regulating gene expression in all organisms. The half-life of mRNA is influenced by its own regulatory cis-acting elements present in the 5Ј-untranslated region, coding region, or 3Ј-untranslated region (22). In general, the stability of mRNA is governed by the interaction of various cytoplasmic/ nuclear proteins (trans-acting factors) with the cis-acting regulatory regions in the mRNA (22). The formation and/or disruption of these RNP complexes in response to various cellular stimuli controls the stability of mRNA. A number of trans-acting factors have been identified and characterized as mRNA-stabilizing, destabilizing, or translational regulatory proteins (23)(24)(25)(26)(27)(28). In the case of LHR mRNA, the interaction of the mRNA-binding protein, LRBP, occurs in the coding region, and the net result of this interaction is a decrease of the cell surface expression of LHR and its transcripts in the ovary.
The present study examined the role of LRBP on the translation of LH receptor mRNA. The decrease in LHR protein translation observed in the presence of partially purified LRBP and not in the presence of an irrelevant protein (bovine serum albumin) indicates a translation inhibitory effect of LRBP. The inhibitory effect was found to be LHR FIGURE 7. Analysis of RNA isolated from immunoprecipitated RNP complex. a, ovaries collected from pseudo-pregnant rats 6 h after hCG injection were homogenized in NET-2 buffer and centrifuged at 10,000 ϫ g for 10 min at 4°C. The supernatant was used to immunoprecipitate the RNP complex using mevalonate kinase antibody. The RNA extracted from the precipitated RNP complex was used for the RT-PCR amplification for detecting the LH receptor mRNA. The generated DNA was then separated on a 0.8% native agarose gel stained with ethidium bromide. Preimmune rabbit IgG and high density lipoprotein-binding protein antibody (an irrelevant antibody) were used as negative controls for the immunoprecipitation. pCMV4 vector containing full-length rat LHR cDNA, and a reaction containing only gene specific primers was used as a positive control and negative control for PCR amplification. kb, kilobases. b, bands B1 and B2 shown in A were isolated and used to amplify the first 300 nt of the rat LHR cDNA as described under "Materials and Methods." The PCR amplification products were then separated on a 0.8% native agarose gel (lanes 1 and 3). pCMV4 vector containing rat LHR cDNA was used as the positive control (lane 5). The amplified bands were then isolated and digested with PstI, and the digested products were separated on native agarose gel stained with ethidium bromide (lanes 2, 4, and 6). mRNA-specific as translation of another, unrelated mRNA (␤-actin) transcript remains unaffected by LRBP. Restoration of translation in the presence of wtLBS and mevalonate indicates that the inhibitory effect of LRBP on LHR mRNA translation was in fact due to the binding of LRBP to the coding region of LHR mRNA. Our previous studies have clearly shown that the interaction of LRBP with LHR mRNA was inhibited by mevalonate and by excess amounts of LBS (12). Thus, the demonstration of the reversal of the translation inhibitory effect of LRBP by mevalonate and the wtLBS underscores their ability to disrupt the formation of ribonucleoprotein complex between LHR mRNA and LRBP. The lack of reversal of the translation inhibition by mLBS, in which all cytidines were mutated to uridine residues, further argues for the exquisite specificity of LRBP to interact with a defined coding region sequence of the LHR mRNA to cause translation inhibition. No appreciable change in mRNA stability in the presence of LRBP in the Flexi rabbit reticulocyte lysate system further confirmed that the inhibition of LHR protein synthesis was in fact due to translation inhibition. The lack of a good in vitro model system for silencing MVK RNA hampered the demonstration of the role of mevalonate kinase as a trans factor in the ligand-induced down-regulation of LH receptor. However, the demonstration of the endogenous association of mevalonate kinase with LH receptor mRNA by immunoprecipitation of LH receptor RNP complex from the ovary using mevalonate kinase antibody (Fig. 7) strongly supports a functional role for mevalonate kinase in receptor down-regulation.
A number of mRNA binding proteins (trans factors) have been implicated in translational regulation of eukaryotic mRNAs. For example, in the case of acid ␤-glucosidase a mammalian cytoplasmic protein, TCP80/NF90, binds to the coding region of ␤-glucosidase mRNA and inhibits its translation (29,30). Similarly heterogeneous nuclear ribonucleoprotein K, poly(pC)-binding protein 1 (PCBP-1), and PCBP-2 bind to the coding region of human Papillomavirus Type 16 L2 mRNA and inhibit its translation in vitro (31). In some cases translation of mRNA is controlled by its own protein, product. For example, human thymidylate synthase and dihydrofolate reductase are regulated in this manner. Thymidylate synthase binds to its own mRNA in the coding region and represses its translation in vitro (27). The existence of thymidylate synthase ribonucleoprotein complex has been shown in human colon cancer cells (32). Thymidylate synthase binds not only to its own mRNA but also binds to the coding regions of c-Myc and p53 mRNAs and inhibits their translation (33,34). Thus, thymidylate synthase functions as an mRNA-binding protein in addition to its role as an enzyme in the synthesis of thymidylate. Human dihydrofolate reductase enzyme has also been shown to bind to its own mRNA and inhibits its translation in vitro (28). Translational repression has also been reported in prokaryotes. For instance, bacteriophage R17 coat protein binds to the translational start site of its own mRNA and represses its translation (35). Similarly, our data presented here demonstrate that mevalonate kinase, in addition to its role as an enzyme in the de novo synthesis of cholesterol, has a dual function of regulating LH receptor mRNA translation.
Translation of eukaryotic mRNA is a complex and highly regulated process. Once transported to the cytoplasm, the fully processed mRNA can exist either in a polysomal-associated, translationally active form or in an inactive form as a ribonucleoprotein (messenger RNP) complex (29,30,36,37). The exchange of mRNA between the polysomal and mRNP compartments modulates the quantity of mRNA available for translation (38). Several studies have implicated a clear link between translation and mRNA stability, since the factors and processes required for mRNA translation and decay are intimately connected (22,36,39). It has been shown that translational arrest in general alters the degradation rate of most eukaryotic mRNAs (22,36,39,40). Several reports have shown that inhibition of translation or aberrant translational termination of some mRNAs destabilizes the transcripts (36,39,41). The mechanism by which translation affects mRNA stability can vary depending on the transcript, cis elements and trans factors and the physiological needs of the cell.
MVK is a member of galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) kinase superfamily of enzymes that are known to have a left-handed ␤-␣-␤ fold, termed the ribosomal protein S5 domain 2-like fold. A similar ␤-␣-␤ fold exists in elongation factor G, ribonuclease P and other RNA/DNA binding proteins (42). Thus, it is conceivable that this fold could be the primary target of MVK interaction with LH receptor mRNA. Although MVK does not posses any intrinsic ribonuclease activity, we have observed an increased degradation of LHR mRNA by LRBP in an in vitro mRNA decay assay using ovarian polysomes (13). In the present study we clearly demonstrate that MVK inhibits LHR mRNA translation and, thus, leads to the formation of an untranslatable mRNP complex. This complex might go into an RNA degradation pathway with the help of other ovarian polysome-associated factors, causing rapid LHR mRNA degradation as we have previously reported (13).
Translational suppression of LHR mRNA by converting it to an untranslatable or less translatable form by interaction with LRBP could regulate the LHR expression both by controlling translation and stability. The formation of an untranslatable LHR mRNP complex instantly reduces the LHR protein synthesis. As a second stage of this translation inhibition, other tissue-specific cytosolic factors capable of recognizing this untranslatable mRNP complex might trigger the decay of LHR mRNA. The ability to regulate the expression of LHR at the translational level might be a quick and efficient way to control receptor expression, especially in response to the constantly changing hormonal milieu during the ovarian cycle.