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The AU-rich element (ARE) is an important instability determinant for a large number of early-response-gene mRNAs. AREs also mediate the stabilization of certain pro-inflammatory mRNAs, such as tumour necrosis factor (TNF)-alpha and cyclo-oxygenase-2 (COX-2), in response to inflammatory stimuli. To understand how AREs control mRNA stability, it is necessary to identify trans-acting factors. We have purified a new ARE-binding protein and identified it as CArG box-binding factor-A (CBF-A). The amino acid sequence of CBF-A is highly similar to that of the ARE-binding protein AUF1. Recombinant CBF-A bound the COX-2 and TNF-alpha AREs, but not a non-specific control RNA. In contrast, in an electrophoretic-mobility-shift assay (EMSA) of crude RAW 264.7 macrophage-like cell extracts, an antiserum that recognizes both AUF1 and CBF-A failed to supershift complexes formed on the TNF-alpha ARE, but did supershift a complex specific for the COX-2 ARE. CBF-A exists as two isoforms, p37 and p42, that differ by a 47-amino-acid insertion close to the C-terminus. By expressing epitope-tagged isoforms of CBF-A it was shown that the p42 isoform binds the COX-2 ARE in EMSA of crude cell extracts. In a HeLa-cell tetracycline-regulated reporter system, overexpression of the p42 CBF-A isoform resulted in stabilization of a COX-2 ARE reporter mRNA. Epitope-tagged p42 CBF-A expressed in HeLa cells co-immunoprecipitated with endogenous COX-2 mRNA, but not glyceraldehyde-3-phosphate dehydrogenase mRNA, as shown by reverse-transcription PCR. The similarity between CBF-A and AUF1 suggests that CBF-A could be re-named AUF2.

Original publication

DOI

10.1042/BJ20020402

Type

Journal

Biochem J

Publication Date

15/09/2002

Volume

366

Pages

709 - 719

Keywords

3' Untranslated Regions, Amino Acid Sequence, Animals, Blotting, Western, Cell Line, Chromatography, Chromatography, High Pressure Liquid, Cyclooxygenase 2, DNA-Binding Proteins, Genes, Reporter, HeLa Cells, Heterogeneous-Nuclear Ribonucleoprotein D, Heterogeneous-Nuclear Ribonucleoprotein Group A-B, Humans, Isoenzymes, Macrophages, Mass Spectrometry, Membrane Proteins, Mice, Molecular Sequence Data, Peptides, Plasmids, Precipitin Tests, Prostaglandin-Endoperoxide Synthases, Protein Binding, Protein Isoforms, RNA, Messenger, RNA-Binding Proteins, Recombinant Proteins, Repressor Proteins, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleases, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Subcellular Fractions, Time Factors, Tumor Necrosis Factor-alpha