title: Two distinct regions in the 3' untranslated region of tumor necrosis factor alpha mRNA form complexes with macrophage proteins.
abstract: The production of tumor necrosis factor alpha (TNF-alpha), a key proinflammatory cytokine essential for the function of the immune system, is regulated at both the transcriptional and posttranscriptional levels. In this report, we focus on the interaction of TNF-alpha mRNA with macrophage proteins, likely mediators of its post-transcriptional control. Mapping of murine TNF-alpha mRNA by using a combination of RNase protection and RNA gel shift assays revealed that two distinct sites within the 3' untranslated region (3'-UTR) engage in the formation of four major RNA-protein complexes, while no protein binding to the 5'-UTR or coding sequences was detected. The protein-binding site of three RNA-protein complexes, A, B, and C, is positioned between bases 1291 and 1320 inside the AU-rich sequence, a region previously shown to be crucial for both translational repression and lipopolysaccharide inducibility of TNF-alpha. An additional protein complex (complex D) whose binding to the TNF-alpha 3'-UTR was independent of the presence of AU-rich sequences was identified. At least six protein species with apparent molecular masses of 48, 52, 54, 81, 101, and 150 kDa are in direct contact with TNF-alpha mRNA. The RNA-binding proteins are differentially distributed in the cell: complexes A and D are present predominantly in the cytosol, while complexes B and C are found in the nucleus and associated with particulate cytoplasmic fractions. Cytosolic complex A displays comparatively high specificity for TNF-alpha mRNA, while the binding of complexes B and C to TNF-alpha mRNA is readily competed for by other AU-rich sequence-containing RNAs. In summary, these findings demonstrate that two regions of the TNF-alpha mRNA molecule interact with macrophage RNA-binding protein complexes that differ in their core protein composition, cellular distribution, and affinity to TNF-alpha mRNA.
authors.affiliation: Centre for the Study of Host Resistance, McGill University, Montreal, General Hospital Research Institute, Quebec, Canada.
authors.forename: Z
authors.initials: Z
authors.lastname: Hel
authors.forename: E
authors.initials: E
authors.lastname: Skamene
authors.forename: D
authors.initials: D
authors.lastname: Radzioch
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009687:Nuclear Proteins / Q000378:metabolism; D014176:Protein Biosynthesis; D015347:RNA Probes; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics*
chemical_list: D009687:Nuclear Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha