PURIFICATION AND PROPERTIES OF A PROTEIN THAT BINDS TO THE C-TERMINALCODING REGION OF HUMAN C-MYC MESSENGER-RNA

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 s uggest that the protein is bound to polysome-associated c-myc mRNA and protects the mRNA from a ribosome-associated endoribonuclease (Bernst ein, P. L., Herrick, D. J., Prokipcak, R. D., and Ross, J. (1992) Gene s and Dev. 6, 642-654). Here, we describe a four-step purification of the binding protein: solubilization from ribo somes, ammonium sulfate precipitation, RNA affinity chromatography, and reverse-phase high per formance liquid chromatography. The 7O-kDa protein can be renatured fr om solutions containing sodium dodecyl sulfate or organic solvents, gr eatly facilitating its purification. Protein binding to c-myc coding r egion RNA is blocked by diamide and N-ethylmaleimide, indicating a req uirement for sulfhydryl groups. The protein also binds to N-myc coding region RNA but with approximately 5-fold lower affinity than to the c omparable c-myc region. Excess c-myc competitor RNA induces 8-fold des tabilization of c-myc mRNA in cell-free mRNA decay extracts. In contra st, N-myc coding region competitor RNA has no effect on c-nye mRNA hal f-life. Therefore, the protein we have purified probably affects c-myc mRNA metabolism with high specificity.