Cytoplasmic protein mRNA interaction mediates cGMP-modulated translational control of the asialoglycoprotein receptor.
Expression of the asialoglycoprotein receptor by the human hepatocellular carcinoma cell line HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level. In a cell-free system, initiation of asialoglycoprotein receptor mRNA translation was dependent on the presence of the 7-methylguanylate cap site and was independent of 8-bromo-cGMP levels in which the cells were grown prior to RNA isolation. Stable transfection of COS-7 cells with deletion constructs of the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region (UTR). Addition of biotin (an activator of guanylate cyclase) induced the expression of beta-galactosidase present as a chimeric plasmid containing the H2b 187-nucleotide 5'-UTR. An RNA gel retardation assay identified a 37-nucleotide cognate sequence within this 187-nucleotide region. Titration of the 5'-UTR with a cytosolic fraction isolated from HuH-7 grown in the presence or absence of 8-bromo-cGMP or biotin provided direct evidence for an RNA-binding protein responsive to intracellular levels of cGMP. Based on these findings, it seems reasonable to propose that reduction of intracellular levels of cGMP by biotin deprivation results in a negative trans-acting factor associating with the 5'-UTR of asialoglycoprotein receptor mRNAs, thereby inhibiting translation.
Stockert
R J
RJ
Department of Medicine, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, New York, New York 10461, USA.
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D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D006151:Guanosine; C016578:7-methylguanosine; C016276:8-bromocyclic GMP; D001710:Biotin; D006152:Cyclic GMP