Posttranscriptional control of renin synthesis: identification of proteins interacting with renin mRNA 3'-untranslated region.
Stabilization and correct localization of mRNA are important features of renin synthesis. To elucidate the molecular basis of cAMP-mediated posttranscriptional control via mRNA stabilization, we analyzed the interaction of human preprorenin (hREN) mRNA 3'-untranslated region (3'-UTR) with proteins of renin synthesizing Calu-6 cells and investigated their functional impact on messenger integrity. To identify hREN mRNA binding proteins, electrophoretic mobility shift assays, UV cross-linking and RNA-affinity chromatography with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were performed. The following six proteins were unambiguously identified as hREN mRNA 3'-UTR binding proteins: hnRNP E1 (synonyms alpha-CP or PCBP), hnRNP K, dynamin, nucleolin, YB-1, and MINT-homologous protein. All proteins contain various RNA binding motifs, and most have been described in the context of mRNA binding and mRNA stabilization. Four proteins for which antibodies were available were verified by immunological techniques (dynamin, nucleolin, hnRNP E1, and YB-1). Forskolin, an activator of cAMP synthesis, considerably stimulates renin synthesis via inhibition of REN mRNA decay. Functionally, this cAMP-based mRNA stabilization is accompanied by a 3- to 6-fold upregulation of REN mRNA binding proteins. RNase degradation assays confirm that 3'-UTR binding proteins are able to protect and stabilize REN mRNA in vitro.
Skalweit
Angela
A
Institut für Physiologie, Humboldt-University Berlin, Charité, Germany.
Doller
Anke
A
Huth
Antje
A
Kähne
Thilo
T
Persson
Pontus B
PB
Thiele
Bernd-Joachim
BJ
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D012083:Renin / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012083:Renin