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.
Lellek
H
H
Medizinische Kernklinik und Poliklinik and the Institut für Zellbiochemie und Klinische Neurobiologie, Universitäts-Krankenhaus Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Kirsten
R
R
Diehl
I
I
Apostel
F
F
Buck
F
F
Greeve
J
J
D000071479:APOBEC-1 Deaminase; D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics* / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003564:Cytidine Deaminase / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014018:Tissue Distribution; D015534:Trans-Activators; D014466:Ultraviolet Rays; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
C411647:A1CF protein, human; D001055:Apolipoproteins B; D018076:DNA, Complementary; C105789:KHSRP protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D000071479:APOBEC-1 Deaminase; C000605089:APOBEC1 protein, human; C000605091:Apobec1 protein, rat; D003564:Cytidine Deaminase