Journal of Lipid Research, Vol 35, 2200-2211, Copyright © 1994 by Lipid Research, Inc.

ARTICLES

Alternative polyadenylation of apolipoprotein B RNA is a major cause of B-48 protein formation in rat hepatoma cell lines transfected with human apoB-100 minigenes

T Heinemann, S Metzger, EA Fisher, JL Breslow and LS Huang
Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, NY 10021.

The human apoB gene encodes an mRNA of 14121 nucleotides. In liver the apoB gene products a full-length mature protein of 4,536 amino acids (B- 100), whereas in the intestine this gene produces a truncated protein of 2,152 amino acids (B-48). B-48 results from RNA editing of nucleotide 6666 from C to U, thereby producing a stop codon at position 2153. Rat liver has been shown to contain apoB RNA editing capability resulting in production of both B-100 and B-48. To create an in vitro expression system for human B-100, a minigene with a wild type coding sequence for the entire B-100 protein (B-100/Gln) was stably transfected into rat hepatoma cells (McA-RH7777). Similarly, a minigene with mutation at nucleotide 6667 that allowed translation even after editing of nucleotide 6666 (B-100/Leu, nonstop mutant), a minigene with an additional nonsense mutation at nucleotide 7053 to produce B-50 (B- 50/Leu), and a truncated wild type minigene with a stop signal at codon 3261 to produce B-74 and an mRNA of 10 kb (B-74/Gln) were also transfected. Very little full-length B-100 and B-74 was produced by any of the respective constructions, including the B-100/Leu with the nonstop mutation. Transfection with B-100/Gln, B-100/Leu and B-74/Gln constructions produced greater than 90% of apoB as B-48, whereas the B- 50/Leu construction produced 76% B-50 and 24% B-48. The inability of the B-100/Leu construction to produce B-100 suggested an explanation for B-48 production other than RNA editing. Northern blot analysis showed that the RNA produced by all four transfectants was shortened to a size of about 7 kb. A 10-kb but no 7-kb RNA was observed in the B- 74/Leu construction when transfected to Chinese hamster ovary cells suggesting cell type specificity in generation of a shortened RNA. The 3' end of apoB RNA from McA-RH7777 B-100/Leu transfectants was reverse transcribed, cloned, and sequenced. This revealed two species of RNA: one polyadenylated at or near nucleotide 6775 capable of coding for B- 48, the other polyadenylated at nucleotide 7080 capable of coding for B- 50. In 18% of the cDNA clones, nucleotide 6666 was edited from C to T. In 6 of 34 clones, addition of the poly(A) tail after nucleotide 6774 created a TAA stop codon, whereas no stop signals could be detected in the remaining clones.(ABSTRACT TRUNCATED AT 400 WORDS)
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