Apolipoprotein (apo) B-100 is a physiological ligand for the low density lipoprotein receptor and its plasma concentration is positively correlated with coronary atherosclerosis. ApoB-48 is an obligatory component of chylomicrons. In humans, it is synthesized in the small intestine. ApoB-48 contain 2152 amino acid residues which correspond to the NH2-terminal 48% of apoB-100. Since the putative receptor-binding domain (s) of apoB-100 resides in the COOH-terminal half of the molecule, apoB-48 lacks this domain and is physiologically an entirely different molecule. ApoB-48 is produced by a recently described highly unique mechanism. The protein was shown to be the product of an intestinal mRNA that has an inframe stop codon UAA resulting from a C--->U change in the codon CAA encoding Gln-2153 in apoB-100 mRNA. The C--->U change appears to be the product of a tissue-specific RNA editing reaction. This application deals with the characterization of the RNA editing reaction in the rat liver. Unlike the human liver which produces almost exclusively apoB-100, the rat liver produces large amounts of apoB-48 as well as apoB-100. Rat liver nuclear extracts contain substantial apoB mRNA editing activity. Preliminary experiments have shown the presence of protein that binds to the editing recognition site on apoB mRNA in a sequence-specific manner. This protein, designated apoB RNA sequence-specific binding protein or BSSBP, has an apparent molecular mass of -40 kDa. It can be crosslinked to apoB RNA by ultraviolet light. The proposed studies include the characterization of the sequence specificity of the binding, the purification of BSSBP, the molecular cloning of a BSSBP cDNA, and the use of the cDNA to examine the developmental regulation of BSSBP mRNA production. The purification of BSSBP will utilize both standard techniques of protein fractionation, including high performance liquid chromatography as well as affinity purification by specific binding to the recognition-site RNA sequence. Identification of cDNA clones will utilize oligonucleotide hybridization, antibody screening of a rat liver expression library and/or specific recognition-site RNA sequence screening of such a library. During all phases of the project, BSSBP activity will be correlated with RNA editing activity. RNA editing is a physiological mechanism whereby the intestine and liver (in the rat) regulate the relative amounts of apoB-48/apoB-100 production. The proposed work will shed light on this process, which also has profound implications for general cell and molecular biology.
| Lau, P P; Zhu, H J; Baldini, A et al. (1994) Dimeric structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene. Proc Natl Acad Sci U S A 91:8522-6 |
| Chan, L (1993) RNA editing: exploring one mode with apolipoprotein B mRNA. Bioessays 15:33-41 |
| Xiong, W; Zsigmond, E; Gotto Jr, A M et al. (1992) Transgenic mice expressing full-length human apolipoprotein B-100. Full-length human apolipoprotein B mRNA is essentially not edited in mouse intestine or liver. J Biol Chem 267:21412-20 |
| Lau, P P; Xiong, W J; Zhu, H J et al. (1991) Apolipoprotein B mRNA editing is an intranuclear event that occurs posttranscriptionally coincident with splicing and polyadenylation. J Biol Chem 266:20550-4 |
