Purification and properties of a protein that binds to the C-terminal coding region of human c-myc mRNA.

Abstract

The short half-life of c-myc mRNA is influenced by sequences in the 3'-untranslated region and the C-terminal part of the coding region. In cell-free extracts, a polysomal protein binds to RNA corresponding to the coding region stability determinant. This and other observations suggest that the protein is bound to polysome-associated c-myc mRNA and protects the mRNA from a ribosome-associated endoribonuclease (Bernstein, P.L., Herrick, D.J., Prokipcak, R.D., and Ross, J. (1992) Genes & Dev. 6, 642-654). Here, we describe a four-step purification of the binding protein: solubilization from ribosomes, ammonium sulfate precipitation, RNA affinity chromatography, and reverse-phase high performance liquid chromatography. The 70-kDa protein can be renatured from solutions containing sodium dodecyl sulfate or organic solvents, greatly facilitating its purification. Protein binding to c-myc coding region RNA is blocked by diamide and N-ethylmaleimide, indicating a requirement for sulfhydryl groups. The protein also binds to N-myc coding region RNA but with approximately 5-fold lower affinity than to the comparable c-myc region. Excess c-myc competitor RNA induces 8-fold destabilization of c-myc mRNA in cell-free mRNA decay extracts. In contrast, N-myc coding region competitor RNA has no effect on c-myc mRNA half-life. Therefore, the protein we have purified probably affects c-myc mRNA metabolism with high specificity.