Apolipoprotein B mRNA editing. Validation of a sensitive assay and developmental biology of RNA editing in the rat.
Apolipoprotein B (apoB) 48 mRNA is the product of a C----U conversion of the first base of the codon CAA for Gln-2153 in apoB-100 mRNA, changing it to an in-frame stop codon (UAA). Methods for measuring the ratio of apoB-48 to apoB-100 mRNA that have been authenticated with standard mixtures of the two apoB mRNA species have not been described. Using RNA mixtures consisting of known proportions of in vitro transcripts of apoB-100 and apoB-48, we directly compared four different assays. We found that a procedure based on the polymerase chain reaction, cDNA cloning, and oligonucleotide colony hybridization was the most sensitive and accurate assay. Total RNAs isolated from adult rat small intestine, adult liver, Day 15 and term placentas, and term fetal membranes were found to contain apoB mRNA in the following relative amounts: 100, 59.8, 0.9, 6.96, and 1087, respectively. They all contained both apoB-48 and apoB-100 mRNAs, with the former constituting 95.8, 59.2, 4.6, 1.3, and 0.8%, respectively, of the apoB mRNA. We examined the ontogeny of apoB-48 mRNA biogenesis in the liver and intestine in the rat prenatally on Days 17, 19, and 20 of gestation, and postnatally on Days 1, 3, 7, 13, 20, 24, and 37 after birth. Slot-blot hybridization demonstrated that apoB mRNA showed a peak at birth (Days 1-3 in the liver and Days 1-7 in the small intestine) and then decreased on Days 7 (in the liver) and 13 (in the intestine) before it increased again on Day 20 toward the adult level. Quantitation of the ratio of apoB-48 to apoB-100 mRNA at the different time points showed that RNA editing became highly competent prenatally on Day 19 of gestation in the small intestine, but postnatally on Day 24 after birth in the liver. The asynchronous nature for this developmentally regulated process in the liver and small intestine of the rat has implications for the mechanism of RNA editing and lipid homeostasis in this animal.
Wu
J H
JH
Department of Cell Biology, Baylor College of Medicine, Houston, Texas.
Semenkovich
C F
CF
Chen
S H
SH
Li
W H
WH
Chan
L
L
D000367:Age Factors; D000818:Animals; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D007421:Intestine, Small / Q000502:physiology; D008099:Liver / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides; D010920:Placenta / Q000502:physiology; D016133:Polymerase Chain Reaction; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats
D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D009841:Oligonucleotides; D012333:RNA, Messenger