Evidence for the association of luteinizing hormone receptor mRNA-binding protein with the translating ribosomes during receptor downregulation

Abstract

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.

Notice of Retraction: The Synthesis of Aspergillus Niger Lipase Gene and the Analysis of Its Translation Initiation Region for the Expression in Pichia pastoris

“…It is generally believed that the existence of a stable mRNA secondary structure at TIR of mRNA is negative for initialing the translation [11,12] , and this structure will affect the combining between ribosome and its ribosome binding site or initiation codons, then interfere with protein expression. Translation initiation would be hindered if the secondary structure is stable too much [13,14] , when its stability decreased, the effect will be weakened and the expression level improved [15,16] . Computational prediction has indicated that reducing mRNA secondary structure can also enhance translation in the yeast expression system.…”

Structure, function and regulation of gonadotropin receptors – A perspective

“…The translocation of LRBP to translating ribosomes was then demonstrated by showingthat this translocation occurs during hCG-induced LHR mRNA downregulation (Menon et al, 2009). Immunoprecipitation of polysomes with antibody against LRBP followed by the analysis of LHR mRNA levels in the immunoprecipitates confirmed that direct interaction of LRBP with LHR mRNA occurs in the ribosomes during downregulation.…”

Polymorphism in the Alternative Donor Site of the Cryptic Exon of LHCGR: Functional Consequences and Associations with Testosterone Level

“…It is conceivable that LHCGR-exon 6A is a non-coding transcript, operating as competing endogenous RNAs (ceRNA) to act as decoys for LHCGR targeting miRNA and to modulate the cellular full-length receptor level2324. The recently reported LHCGR binding protein (LRBP), also known as mevalonate kinase, could associate with LHCGR mRNA to form an untranslatable ribonucleoprotein complex and thus inhibit LHCGR mRNA translation2526. The interaction between LRBP and LHCGR-exon 6A deserves further investigation.…”