title: Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary.
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.
authors.affiliation: Department of Obstetrics/Gynecology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0617, USA.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Nair
authors.forename: K M J
authors.initials: KM
authors.lastname: Menon
mesh_terms: D000818:Animals; D003562:Cytidine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005786:Gene Expression Regulation; D047468:Immunoprecipitation; D008798:Mevalonic Acid / Q000737:chemistry; D009154:Mutation; D010053:Ovary / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011974:Receptors, LH / Q000096:biosynthesis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011974:Receptors, LH; D003562:Cytidine; D012313:RNA; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D008798:Mevalonic Acid