An mRNA Stability Complex Functions with Poly(A)-Binding Protein To Stabilize mRNA In Vitro

Abstract

The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. α-Globin mRNA stability is dictated by sequences in the 3′ untranslated region (3′UTR) which form a specific ribonucleoprotein complex (α-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, αCP1 and αCP2. Using an in vitro-transcribed and polyadenylated α-globin 3′UTR, we have devised an in vitro mRNA decay assay which reproduces the α-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the α-complex by sequestration of αCP1 and αCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the α-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between αCP1 and αCP2 with PABP suggests that the α-complex can directly interact with PABP. Therefore, the α-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.

ACKNOWLEDGMENTS

We thank K. Novick for excellent technical assistance, G. Dreyfuss and M. Siomi for providing the PABP clone and antibody, S. Gunderson for providing the bPAP expression plasmid bPAP-423 and the U1A expression plasmid pET-U1A, S. Liebhaber for plasmids pSV2A-α2H9 and pSV2A-α2H21, and S. Peltz for the vaccinia virus capping enzyme. We also thank S. Gunderson, J. Huibregtse, and S. Peltz for critical reading of the manuscript.

This work was supported by funds from Rutgers University and National Institutes of Health grant DK51611 to M.K. N.D. was supported by an American Heart Association predoctoral fellowship.