Purification and properties of a protein that binds to the C-terminal coding region of human c-myc mRNA.
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.
Prokipcak
R D
RD
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Herrick
D J
DJ
Ross
J
J
D001665:Binding Sites; D001667:Binding, Competitive; D005786:Gene Expression Regulation; D016259:Genes, myc; D006801:Humans; D008970:Molecular Weight; D011132:Polyribosomes / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D013439:Sulfhydryl Reagents