Cyclic nucleotide regulation of PAI-1 mRNA stability. Identification of cytosolic proteins that interact with an a-rich sequence.
Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator-inhibitor (PAI-1) mRNA. Previous studies utilizing HTC cells stably transfected with beta-globin:PAI-1 chimeric constructs demonstrated that at least two regions within the PAI-1 3'-untranslated region mediate the cyclic nucleotide-induced destabilization of PAI-1 mRNA; one of these regions is the 3'-most 134 nucleotides (nt) of the PAI-1 mRNA (Heaton, J. H., Tillmann-Bogush, M., Leff, N. S., and Gelehrter, T. D. (1998) J. Biol. Chem. 273, 14261-14268). In the present study, ultraviolet cross-linking analyses of this region demonstrate HTC cell cytosolic mRNA-binding proteins ranging from 38 to 76 kDa, with a major complex migrating at approximately 50 kDa. RNA electrophoretic mobility shift analyses demonstrate high molecular weight multiprotein complexes that specifically interact with the 134-nt cyclic nucleotide-responsive sequence. The 50, 61, and 76 kDa and multiprotein complexes form with an A-rich sequence at the 3' end of the cyclic nucleotide-responsive region; a 38-kDa complex forms with a U-rich region at the 5' end of the 134 nt sequence. Mutation of the A-rich region prevents both the binding of the 50-, 61-, and 76-kDa proteins and formation of the multiprotein complexes, as well as cyclic nucleotide-regulated degradation of chimeric globin:PAI-1 transcripts in HTC cells. These data suggest that the proteins identified in this report play an important role in the cyclic nucleotide regulation of PAI-1 mRNA stability.
Tillmann-Bogush
M
M
Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618, USA.
Heaton
J H
JH
Gelehrter
T D
TD
D020413:3' Untranslated Regions; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D003600:Cytosol / Q000378:metabolism*; D016296:Mutagenesis; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000187:drug effects* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate