- Menon, Bindu; Peegel, Helle; Menon, K M
- Luteinizing hormone receptor (LHR) mRNA is post-transcriptionally regulated during ligand-induced downregulation. This process involves interaction of LHR mRNA with a specific mRNA-binding protein (LRBP), identified as mevalonate kinase (MVK), resulting in inhibition of translation followed by targeting the ribonucleoprotein complex to accelerated degradation. The present study investigated the endogenous association of LRBP with the translational machinery and its interaction with LHR mRNA during LH/hCG-induced downregulation. Ovaries were collected from rats that were injected with the ligand, hCG, to induce downregulation of LHR mRNA expression. Western blot analysis showed significantly higher levels of LRBP in polysomes from downregulated ovaries compared to controls. Western blot analysis of ribosome-rich fractions from FPLC-assisted gel filtration of post-mitochondrial supernatants confirmed the presence of LRBP in translating ribosomes isolated from the downregulated state but not from controls. The association of LRBP with LHR mRNA in the downregulated polysomes was demonstrated by immunoprecipitation with LRBP antibody followed by qPCR analysis of the associated RNA. Increased association of LHR mRNA with LRBP during downregulation was also demonstrated by subjecting the polysome-associated RNAs to oligo(dT) cellulose chromatography followed by immunoprecipitation and qPCR analysis. Additionally, analysis of in vitro translation of LHR mRNA showed increased inhibition of translation by polysomes from downregulated ovaries compared to control. This study provides strong in vivo and in vitro evidence to show that during ligand-induced downregulation, LRBP translocates to ribosomes and associates with LHR mRNA to form an untranslatable ribonucleoprotein complex and inhibits LHR mRNA translation, paving the way to its degradation.
- Biochimica et biophysica acta Journal
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