Identification and cDNA cloning of a novel RNA-binding protein that interacts with the cyclic nucleotide-responsive sequence in the type-1 plasminogen activator inhibitor mRNA

Incubation of HTC rat hepatoma cells with 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator inhibit or (PAI-1) mRNA. We have reported previously that the 3'-most 134 nt of the PAI-1 mRNA is able to confer cyclic nucleotide regulation of message stabi lity onto a heterologous transcript. R-EMSA and UV cross-linking experiment s have shown that this 134 nt cyclic nucleotide-responsive sequence (CRS) b inds HTC cell cytoplasmic proteins ranging in size from 38 to 76 kDa. Mutat ions in the A-rich region of the CRS both eliminate cyclic nucleotide regul ation of mRNA decay and abolish RN-protein complex formation, suggesting th at these RNA-binding proteins may be important regulators of mRNA stability . By sequential R-EMSA and SOS-PAGE we have purified a protein from HTC cel l polysomes that binds to the PAI-1 CRS. N-terminal sequence analysis and a search of protein data bases revealed identity with two human sequences of unknown function. We have expressed one of these sequences in E. coli and confirmed that the recombinant protein interacts specifically with the PAI- 1 CRS, Mutation of the A-rich portion of the PAI-1 CRS reduces binding by t he recombinant PAI-1 RNA-binding protein. The amino acid sequence of this p rotein includes an RGG box and two arginine-rich regions, but does not incl ude other recognizable RNA binding motifs, Detailed analyses of nucleic aci d and protein data bases demonstrate that blocks of this sequence are highl y conserved in a number of metazoans, including Arabidopsis, Drosophila, bi rds, and mammals. Thus, we have described a novel RNA-binding protein that identifies a family of proteins with a previously undefined sequence motif. Our results suggest that this protein, PAI-RBP1, may play a role in regula tion of mRNA stability.