LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5'-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.
Supplementation of compost at casing with various ground seeds caused greater increases in mushroom yield than their respective seed oil meals when supplemented and compared at equivalent rates of nitrogen addition.Supplementation with various refined and crude seed oils increased mushroom yield, particularly in the first break or flush of mushrooms. This constitutes evidence for a relationship between lipid metabolism and the initiation of fruiting in the cultivated mushroom, Agaricus bisporus (Lange) Sing.
Luteinizing hormone receptor (LHR) undergoes downregulation during preovulatory LH surge through a post-transcriptional mechanism involving an RNA binding protein designated as LRBP. The present study examined the mechanism by which LRBP induces the degradation of LHR mRNA, specifically the role of decapping of LHR mRNA and the translocation of LRBP-bound LHR mRNA to degradative machinery. Immunoprecipitation of the complex with the 5’cap structure antibody followed by real time PCR analysis showed progressive loss of capped LHR mRNA during downregulation suggesting that LHR mRNA undergoes decapping prior to degradation. RNA immunoprecipitation (RIP) analysis confirmed dissociation of eIF4E from the cap structure, a step required for decapping. Furthermore, RIP analysis using antibody against the p body marker protein, DCP1A showed that LHR mRNA was associated with the p bodies, the cytoplasmic foci that contains RNA degradative enzymes and decapping complex. Immunohistochemical studies using antibodies against LRBP and DCP1A followed by confocal analysis showed colocalization of LRBP with DCP1A during downregulation. This was further confirmed by co-immunoprecipitation of LRBP with DCP1A. The association of LRBP and LHR mRNA in the p bodies during downregulation was further confirmed by examining the association of a second p body component, rck/p54, using IP and RIP, respectively. These data suggest that the association of LRBP with LHR mRNA results in the translocation of the mRNP complex to the p bodies leading to decapping and degradation.
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