BINDING OF A 50-KD PROTEIN TO A U-RICH SEQUENCE IN AN MESSENGER-RNA ENCODING A PROLINE-RICH PROTEIN THAT IS DESTABILIZED BY FUNGAL ELICITOR

The mRNA encoding the bean proline-rich protein PvPRP1 has been shown previously to be destabilized in elicitor-treated cells. In this study , we identified a 50-kD protein in cellular extracts that binds specif ically to the PvPRP1 mRNA by UV cross-linking assays. Using P-32-label ed RNAs transcribed in vitro from a series of 5' deleted PvPRP1 cDNA c lones, we demonstrated that the PvPRP1 mRNA binding protein (PRP-BP) b inds to a 27-nucleotide U-rich (similar to 60%) domain in the 3' untra nslated region. Poly(U) and, to a lesser extent, poly(A-U) competed fo r the PRP-BP binding activity. PRP-BP activity is redox regulated in v itro, as shown by the effects of sulfhydryl-modifying reagents on the RNA binding activity. Treatment of cellular extracts with the reducing agents DTT and beta-mercaptoethanol increased binding activity, where as treatment with the oxidizing agent diamide and the alkylating agent N-ethylmaleimide inhibited binding. In extracts from elicitor-treated cells, PRP-BP activity increased approximately fivefold prior to rapi d PvPRP1 mRNA degradation. The increase in PRP-BP activity was apparen tly due to post-translational regulation because control and elicitor- treated cell extracts supplemented with DTT showed high comparable lev els of RNA binding activity. The kinetics of PRP-BP activation after e licitor treatment and its capacity for redox regulation in vitro sugge sted that PRP-BP may function in the elicitor-induced destabilization of PvPRP1 mRNA.