Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex

J Biol Chem. 2000 Jun 30;275(26):19848-56. doi: 10.1074/jbc.M001786200.

Abstract

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AICDA (Activation-Induced Cytidine Deaminase)
  • APOBEC-1 Deaminase
  • Amino Acid Sequence
  • Animals
  • Apolipoproteins B / genetics*
  • Apolipoproteins B / isolation & purification*
  • Chromatography, Affinity
  • Chromatography, Gel
  • Cytidine Deaminase / metabolism
  • DNA, Complementary / metabolism
  • Humans
  • Liver / metabolism
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA Processing, Post-Transcriptional
  • RNA, Messenger / genetics*
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / isolation & purification*
  • RNA-Binding Proteins / metabolism
  • Rats
  • Recombinant Proteins / metabolism
  • Tissue Distribution
  • Trans-Activators*
  • Ultraviolet Rays

Substances

  • A1CF protein, human
  • Apolipoproteins B
  • DNA, Complementary
  • KHSRP protein, human
  • RNA, Messenger
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Trans-Activators
  • AICDA (Activation-Induced Cytidine Deaminase)
  • APOBEC-1 Deaminase
  • APOBEC1 protein, human
  • Apobec1 protein, rat
  • Cytidine Deaminase

Associated data

  • GENBANK/AJ272078
  • GENBANK/AJ272079