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.
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)
Heinemann
T
T
Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, NY 10021.
Metzger
S
S
Fisher
E A
EA
Breslow
J L
JL
Huang
L S
LS
D000818:Animals; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017393:RNA Editing; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D014162:Transfection
D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D012333:RNA, Messenger; D011061:Poly A