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 de monstrated that at least two regions within the PAI-1 3'-untranslated regio n 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 (Hea ton, 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-link ing analyses of this region demonstrate HTC cell cytosolic mRNA-binding pro teins ranging from 38 to 76 kDa, with a major complex migrating at similar to 50 kDa. RNA electrophoretic mobility shift analyses demonstrate high mol ecular weight multiprotein complexes that specifically interact with the 13 4-nt cyclic nucleotide-responsive sequence. The 50, 61, and 76 kDa and mult iprotein 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 prevent s both the binding of the 50-, 61-, and 76-bDa proteins and formation of th e multiprotein complexes, as well as cyclic nucleotide-regulated degradatio n of chimeric globin:PAI-1 transcripts in HTC cells. These data suggest tha t the proteins identified in this report play an important role in the cycl ic nucleotide regulation of PAI-1 mRNA stability.