Apolipoprotein B mRNA editing is an intranuclear event that occurs posttranscriptionally coincident with splicing and polyadenylation.
The subcellular compartment in which apolipoprotein (apo) B mRNA is edited is unknown. We studied the site of endogenous apoB mRNA editing and correlated the extent of editing with mRNA maturation in the rat liver. RNA editing activity was demonstrated in both nuclear and cytoplasmic extracts. The specific activity of the editing activity was 5.5-fold higher in the nuclear extract, which was not accounted for by activators, inhibitors, or modulators. However, the total editing activity was 3.1 times higher in the cytoplasmic extract. Highly purified rat liver nuclear apoB mRNA contained 17.3 +/- 1.45% edited sequences compared with 56 +/- 2.5% and 62.15 +/- 6.2% edited sequences in hepatic total and polysomal RNAs, respectively. Because of the significant extent of editing of total nuclear RNA, we fractionated it into a poly(A-) and poly(A+) fraction. While the poly(A-) nuclear fraction contained only 10.4 +/- 1.1% edited sequences, which represents a maximum estimate, the poly(A+) nuclear apoB mRNA contained 50 +/- 1.8% edited sequences, a value very similar to that for polysomal RNA. By direct sequencing of cDNA and genomic clones, we found that as in the case of the human apoB gene, the rat apoB gene contains an intron 25 immediately upstream of the edited exon 26. Using this information, we developed a method to examine in a highly selective manner apoB mRNA that is present in the nucleus before splicing of intron 25 and after splicing of this intron. The unspliced nuclear pre-mRNA contained 7.4 +/- 0.2% edited sequences compared with 51.0 +/- 0.9% edited sequences in the spliced nuclear apoB mRNA. Furthermore, in the poly(A-) pool of apoB pre-mRNA, unspliced nuclear pre-mRNA contained hardly any (1.56%) edited sequences, and the spliced nuclear pre-mRNA contained 7.8 +/- 0.6% edited mRNA. In the poly(A+) fraction, unspliced nuclear pre-mRNA had 25.4 +/- 0.05% and spliced nuclear mRNA 53 +/- 0.6% of its apoB mRNA in an edited form. We conclude that in the rat liver apoB mRNA editing is not a cotransciptional event. It occurs posttranscriptionally, but the process is essentially complete in the spliced polyadenylated apoB mRNA before it leaves the nucleus. Little, if any, additional editing occurs in the cytoplasmic compartment.
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
P P
PP
Lau
W J
WJ
Xiong
H J
HJ
Zhu
S H
SH
Chen
L
L
Chan
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains
D001055:Apolipoproteins B; D012333:RNA, Messenger; D011061:Poly A