Polysome-associated c-tnyc mRNA is degraded relatively rapidly in cells and in an in vitro mRNA decay system containing extracts from cultured mammalian cells. Using this system, a competition/screening assay was devised to search for factors that bind to specific regions of polysome-associated c-myc mRNA and thereby alter its half-life. mRNA stability was first assayed in reactions containing exogenous competitor RNAs corresponding to portions of c-myc mRNA itself. The addition of a 182-nucleotide sense strand fragment from the carboxy-terminal portion of the c-myc-coding region destabilized c-myc mRNA by at least eightfold. This RNA fragment had no effect on the stability of other mRNAs tested. Moreover, c-myc mRNA was not destabilized in reactions containing unrelated competitor RNAs or sense strand RNA from the c-myc 5' region. Polysome-associated globin mRNA containing the c-myc-coding region segment in-frame was also destabilized in vitro by the 182-nucleotide RNA. As determined by UV-cross-linking experiments, the 182-nucleotide RNA fragment was recognized by and bound to an ~75-kD polysome-associated protein. On the basis of these data plus Northern blotting analyses of c-myc mRNA decay products, we suggest that the ~75-kD protein is normally bound to a c-myc-coding region determinant and protects that region of the mRNA from endonuclease attack. Possible links between the protective protein, translation, ribosome pausing, and c-myc mRNA turnover are discussed.

The half-life of c-myc mRNA is regulated when cells change their growth rates or differentiate. Two regions within c-myc mRNA determine its short half-life. One is in the 3′-untranslated region, the other is in the coding region. A cytoplasmic protein, the coding region determinant-binding protein (CRD-BP), binds in vitro to the c-myc coding region instability determinant. We have proposed that the CRD-BP, when bound to the mRNA, shields the mRNA from endonucleolytic attack and thereby prolongs the mRNA half-life. Here we report the cloning and further characterization of the mouse CRD-BP, a 577 amino acid protein containing four hnRNP K-homology domains, two RNP domains, an RGG RNA-binding domain and nuclear import and export signals. The CRD-BP is closely related to the chicken β-actin zipcode-binding protein and is similar to three other proteins, one of which is overexpressed in some human cancers. Recombinant mouse CRD-BP binds specifically to c-myc CRD RNA in vitro and reacts with antibody against human CRD-BP. Most of the CRD-BP in the cell is cytoplasmic and co-sediments with ribosomal subunits.

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