An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro

The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. alpha-globin mRNA stability is dictated by sequences in the 3' untranslated region (3'UTR) which form a specific ri bonucleoprotein complex (alpha-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a fa mily of two polycytidylate-binding proteins, alpha CP1 and alpha CP2. Using an in vitro-transcribed and polyadenylated alpha-globin 3'UTR, we have dev ised an in vitro mRNA decay assay which reproduces the alpha-complex-depend ent mRNA stability observed in cells, Incubation of the RNA with erythroleu kemia K562 cytosolic extract results in deadenylation with distinct interme diates containing a periodicity of approximately 30 nucleotides, which is c onsistent with the binding of poly(A)-binding protein (PABP) monomers. Disr uption of the alpha-complex by sequestration of alpha CP1 and alpha CP2 enh ances deadenylation and decay of the mRNA, while reconstitution of the alph a-complex stabilizes the mRNA. Similarly, PABP is also essential for the st ability of mRNA in vitro, since rapid deadenylation resulted upon its deple tion. An RNA-dependent interaction between alpha CP1 and alpha CP2 with PAB P suggests that the alpha-complex can directly interact with PABP. Therefor e, the a-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.