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

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.
Mesh Terms:
Amino Acid Sequence, Animals, Apolipoproteins B, Chromatography, Affinity, Chromatography, Gel, Cytidine Deaminase, DNA, Complementary, Humans, Liver, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, RNA Processing, Post-Transcriptional, RNA, Messenger, RNA-Binding Proteins, Rats, Recombinant Proteins, Tissue Distribution, Trans-Activators, Ultraviolet Rays
J. Biol. Chem.
Date: Jun. 30, 2000
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