Identification of a novel AU-rich-element-binding protein which is related to AUF1

Biochem J. 2002 Sep 15;366(Pt 3):709-19. doi: 10.1042/BJ20020402.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Cell Line
  • Chromatography
  • Chromatography, High Pressure Liquid
  • Cyclooxygenase 2
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Genes, Reporter
  • HeLa Cells
  • Heterogeneous Nuclear Ribonucleoprotein D0
  • Heterogeneous-Nuclear Ribonucleoprotein D*
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B*
  • Humans
  • Isoenzymes / metabolism
  • Macrophages / metabolism
  • Mass Spectrometry
  • Membrane Proteins
  • Mice
  • Molecular Sequence Data
  • Peptides / chemistry
  • Plasmids / metabolism
  • Precipitin Tests
  • Prostaglandin-Endoperoxide Synthases / metabolism
  • Protein Binding
  • Protein Isoforms
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / chemistry*
  • Recombinant Proteins / metabolism
  • Repressor Proteins / chemistry*
  • Repressor Proteins / genetics*
  • Repressor Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Ribonucleases / metabolism
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Subcellular Fractions / metabolism
  • Time Factors
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • 3' Untranslated Regions
  • DNA-Binding Proteins
  • HNRNPD protein, human
  • Heterogeneous Nuclear Ribonucleoprotein D0
  • Heterogeneous-Nuclear Ribonucleoprotein D
  • Heterogeneous-Nuclear Ribonucleoprotein Group A-B
  • Hnrpd protein, mouse
  • Isoenzymes
  • Membrane Proteins
  • Peptides
  • Protein Isoforms
  • RNA, Messenger
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Repressor Proteins
  • Tumor Necrosis Factor-alpha
  • Hnrnpab protein, mouse
  • Cyclooxygenase 2
  • PTGS2 protein, human
  • Prostaglandin-Endoperoxide Synthases
  • Ribonucleases

Associated data

  • GENBANK/AY137376