HADHB, HuR, and CP1 bind to the distal 3'-untranslated region of human renin mRNA and differentially modulate renin expression.
Production of renin is critically dependent on modulation of REN mRNA stability. Here we sought to elucidate the molecular mechanisms involved. Transfections of renin-expressing Calu-6 cells with reporter constructs showed that a cis-acting 34-nucleotide AU-rich "renin stability regulatory element" in the REN 3'-untranslated region (3'-UTR) contributes to basal REN mRNA instability. Yeast three-hybrid screening with the REN 3'-UTR as bait isolated HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) beta-subunit) as a novel REN mRNA-binding protein. Recombinant HADHB bound specifically to the 3'-UTR of REN mRNA, as did the known mRNA stabilizers HuR and CP1 (poly(C)-binding protein-1). This required the renin stability regulatory element. Forskolin, which augments REN mRNA stability in Calu-6 cells, increased binding of several proteins, including HuR and CP1, to the REN 3'-UTR, whereas 4-bromocrotonic acid, a specific thiolase inhibitor, decreased binding and elevated renin protein levels. Upon decreasing HADHB mRNA with RNA interference, renin protein and mRNA stability increased, whereas RNA interference against HuR caused these to decrease. Immunoprecipitation and reverse transcription-PCR of Calu-6 extracts confirmed that HADHB, HuR, and CP1 each associate with REN mRNA in vivo. Intracellular imaging revealed distinct localization of HADHB to mitochondria, HuR to nuclei, and CP1 throughout the cell. Immunohistochemistry demonstrated enrichment of HADHB in renin-producing renal juxtaglomerular cells. In conclusion, HADHB, HuR, and CP1 are novel REN mRNA-binding proteins that target a cis-element in the 3'-UTR of REN mRNA and regulate renin production. cAMP-mediated increased REN mRNA stability may involve stimulation of HuR and CP1, whereas REN mRNA decay may involve thiolase-dependent pathways.
Adams
David J
DJ
Basic & Clinical Genomics Laboratory, School of Medical Sciences and Institute for Biomedical Research, The University of Sydney, New South Wales 2006, Australia.
Beveridge
Dianne J
DJ
van der Weyden
Louise
L
Mangs
Helena
H
Leedman
Peter J
PJ
Morris
Brian J
BJ
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism*; D000230:Adenocarcinoma / Q000378:metabolism; D000818:Animals; D000954:Antigens, Surface; D001483:Base Sequence; D001665:Binding Sites; D005576:Colforsin / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D007668:Kidney / Q000201:enzymology; D008175:Lung Neoplasms; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D063991:Mitochondrial Trifunctional Protein; D063993:Mitochondrial Trifunctional Protein, beta Subunit; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D012083:Renin / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009097:Multienzyme Complexes; C094604:PCBP1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D005576:Colforsin; C576206:HADHB protein, human; D063991:Mitochondrial Trifunctional Protein; D063993:Mitochondrial Trifunctional Protein, beta Subunit; D012083:Renin