The Poly(A)-Binding Protein and an mRNA Stability Protein Jointly Regulate an Endoribonuclease Activity

Abstract

We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable α-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated α-globin 3′ untranslated region (3′UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3′UTR was inefficiently 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 the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated α-globin 3′UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the αCP mRNA stability protein. Sequestration of αCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of αCP to the α-globin 3′UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by αCP, implying that a stable higher-order structural network is involved in stabilization of the α-globin mRNA. Upon deadenylation, the interaction of PABP with αCP would be disrupted, rendering the α-globin 3′UTR more susceptible to endoribonuclease cleavage. The data demonstrated 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.

ACKNOWLEDGMENTS

We thank N. D. Rodgers and P. Trifillis for helpful discussions and critical reading of the manuscript and N. Shanmugam for providing the hnRNP U RNA-binding domain protein.

This work was supported by funds from the National Institutes of Health (grant DK51611) to M.K.