Protein kinase A stimulates binding of multiple proteins to a U-rich domain in the 3'-untranslated region of lactate dehydrogenase A mRNA that is required for the regulation of mRNA stability.
We have explored the molecular basis of the cAMP-induced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasmic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-nucleotide uridine-rich sequence in the LDH 3'-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that specific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stranded stem structure, thus indicating a high degree of primary and secondary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and contains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. The 3'-untranslated region binding activity of the proteins is up-regulated after protein kinase A activation, whereas protein dephosphorylation is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and CSR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processing pathway that specifically stabilizes LDH mRNA in response to activation of the protein kinase A signal transduction pathway.
Department of Cellular and Molecular Biology and Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.
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Tian
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Huang
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Brown
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Jungmann
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology*; D003857:Deoxyuridine / Q000378:metabolism; D006207:Half-Life; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014466:Ultraviolet Rays
D020413:3' Untranslated Regions; D016601:RNA-Binding Proteins; D000242:Cyclic AMP; D007770:L-Lactate Dehydrogenase; D017868:Cyclic AMP-Dependent Protein Kinases; D003857:Deoxyuridine