A 40 kilodalton rat liver nuclear protein binds specifically to apolipoprotein B mRNA around the RNA editing site.
Apolipoprotein (apo) B-48 mRNA is the product of RNA editing which consists of a C----U conversion changing a CAA codon encoding Gln-2153 in apoB-100 mRNA to a UAA stop codon in apoB-48 mRNA. In the adult rat, RNA editing occurs both in the small intestine and the liver. We have studied the ability of rat liver nuclear extracts to bind to synthetic apoB mRNA segments spanning the editing site. Using an RNA gel mobility shift assay, we found the sequence-specific binding of a protein(s) to a 65-nucleotide apoB-100 mRNA. UV crosslinking followed by T1 ribonuclease digestion and SDS-polyacrylamide gel electrophoresis demonstrated the formation of a 40 kDa protein-RNA complex when 32P-labeled apoB-100 mRNA was incubated with a rat liver nuclear extract but not with HeLa nuclear extract. Binding was specific for the sense strand of apoB mRNA, and was not demonstrated with single-stranded apoB DNA, or antisense apoB RNA. The complex also failed to form if SDS was present during the UV light exposure. Binding experiments using synthetic apoB mRNAs indicate that the 40 kDa protein would also bind to apoB-48 mRNA but not apoA-I, apoA-IV, apoC-II or apoE mRNA. Experiments using deletion mutants of apoB-100 mRNA indicate efficient binding of wildtype 65-nucleotide (W65), 40-nucleotide (W40) and 26-nucleotide (W26) apoB-100 mRNA segments, but not 10-nucleotide (or smaller) segments of apoB-100 mRNA to the 40 kDa protein. In contrast, two other regions of apoB-100 mRNA, B-5' (bases 1128-3003) and B-3' (bases 11310-11390), failed to bind to the protein. The 40 kDa sequence-specific binding protein in rat liver nuclear extract may play a role in apoB-100 mRNA editing.
Lau
P P
PP
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Chen
S H
SH
Wang
J C
JC
Chan
L
L
D000818:Animals; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D016372:RNA, Antisense / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D009687:Nuclear Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger