A 32-kilodalton protein binds to AU-rich domains in the 3' untranslated regions of rapidly degraded mRNAs.
An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated region of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of beta-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of beta-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life.
Vakalopoulou
E
E
Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, New Jersey 08544.
Schaack
J
J
Shenk
T
T
D000225:Adenine; D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005914:Globins / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D006367:HeLa Cells / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011519:Proto-Oncogenes; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D014158:Transcription, Genetic; D014162:Transfection; D014498:Uracil
D002352:Carrier Proteins; D009687:Nuclear Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014498:Uracil; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005914:Globins; D000225:Adenine