The poly(A)-binding protein and an mRNA stability protein jointly regulatean endoribonuclease activity

We previously identified a sequence-specific erythroid cell-enriched endori bonuclease (ErEN) activity involved in the turnover of the stable alpha-glo bin mRNA, We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated alpha-globin 3' untranslated region (3'UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3'UTR was i nefficiently cleaved in an in vitro decay assay. The influence of the poly( A) tail was mediated through the poly(A)-binding protein (PABP) bound to th e poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenyl ated alpha-globin 3'UTR was accentuated upon depletion of PABP from the cyt osolic extract, while addition of recombinant PABP reestablished the inhibi tion of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the alpha CP mRNA stability protein. Sequestrat ion of alpha CP resulted in an increase of ErEN cleavage activity, regardle ss of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of alpha CP to the alpha-globin 3 'UTR, which in turn protected the ErEN target sequenc e. Conversely, the binding of PABP to the poly(A) tail was also augmented b y alpha CP, implying that a stable higher-order structural network is invol ved in stabilization of the alpha-globin mRNA, Upon deadenylation, the inte raction of PABP with alpha CP would be disrupted, rendering the alpha-globi n 3'UTR more susceptible to endoribonuclease cleavage. The data demonstrate d a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.