ALTERNATIVE POLYADENYLATION OF APOLIPOPROTEIN-B RNA IS A MAJOR CAUSE OF B-48 PROTEIN-FORMATION IN RAT HEPATOMA-CELL LINES TRANSFECTED WITH HUMAN APO-B-100 MINIGENES

The human apoB gene encodes an mRNA of 14121 nucleotides. In liver the apoB gene produces a full-length mature protein of 4,536 amino acids (B-100), whereas in the intestine this gene produces a truncated prote in of 2,152 amino acids (B-48). B-48 results from RNA editing of nucle otide 6666 from C to U, thereby producing a stop codon at position 215 3. Rat liver has been shown to contain apoB RNA editing capability res ulting in production of both B-100 and B-48. To create an in vitro exp ression system for human B-100, a minigene with a wild type coding seq uence for the entire B-100 protein (B-100/Gln) was stably transfected into rat hepatoma cells (McA-RH7777). Similarly, a minigene with mutat ion at nucleotide 6667 that allowed translation even after editing of nucleotide 6666 (B-100/Leu, nonstop mutant), a minigene with an additi onal 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. V ery little full-length B-100 and B-74: was produced by any of the resp ective constructions, including the B-100/Leu with the nonstop mutatio n. Transfection with B-100/Gln, B-100/Leu and B-74/Gln constructions p roduced greater than 90% of apoB as B-48, whereas the B-50/Leu constru ction produced 76% B-50 and 24% B-48. The inability of the B-100/Leu c onstruction to produce B-100 suggested an explanation for B-48 product ion 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 IO-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 f rom 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 polya denylated 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 clone s, addition of the poly(A) tail after nucleotide 6774 created a TAA st op codon, whereas no stop signals could be detected in the remaining c lones. These studies suggest that lack of human B-100 expression in Mc A-RH7777 cells is due to alternative polyadenylation of apoB RNA at cr yptic polyadenylation sites.