Abstract
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.
Highlights
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]
Previous studies from our laboratory have shown that the decline in cell surface expression of ovarian Luteinizing hormone receptor (LHR) seen after human chorionic gonadotropin (hCG) administration is paralleled by a specific, transient loss of all four LHR mRNA transcripts [10]
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.”
Summary
Biological actions of luteinizing hormone (LH), 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]. 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]. 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]. 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
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