Apolipoprotein B48 RNA editing in chimeric apolipoprotein EB mRNA.
Apolipoprotein (apo) B occurs in two forms, apoB100 (512 kDa) and apoB48 (240 kDa); both are derived from the same gene. A novel mechanism involving editing of the apoB mRNA causes the formation of apoB48; the first base of codon 2153 is changed from cytosine to uracil, converting a glutamine codon to a premature stop codon. To identify the apoB mRNA sequence elements recognized by the apoB mRNA editing mechanism, two apoB cDNA fragments (354 and 63 base pairs) with codon 2153 near their centers were inserted into a high expression vector of another secreted apolipoprotein, apoE. The resulting vectors, pHEB-354 and -63, were transfected into Chinese hamster ovary cells, HepG2 cells, and apoB48-producing CaCo-2 cells. The secreted chimeric apolipoproteins (apoEB354 and apoEB63) were analyzed for premature truncation, and the mRNA was analyzed for the presence of an edited base. The pHEB-354 construct produced a truncated protein only in CaCo-2 cells, whereas pHEB-63 produced no truncated protein in any of the three cell types. The mRNA was converted to cDNA and amplified by the polymerase chain reaction technique. Differential hybridization of the polymerase chain reaction products with CAA (Gln) and TAA (Stop) specific probes detected an edited base only in cDNA from CaCo-2 cells transfected with pHEB-354, in agreement with the protein analysis. We conclude that the nucleotide sequence of the apoB cDNA insert in pHEB-354 contains sufficient information to be edited in CaCo-2 cells. In these cells, a cryptic polyadenylation site was activated in the edited pHEB-354 mRNA. As a result, CaCo-2 cells transfected with pHEB-354 produced a short, edited pHEB-354 mRNA and a long, unedited pHEB-354 mRNA. Chinese hamster ovary cells transfected with pHEB-354 or CaCo-2 cells transfected with pHEB-63 produced only a full length transcript. Amplification of the pHEB-354 cDNA using 3'-primers upstream and downstream of the poly(A) addition site and hybridization with the TAA probe confirmed these results. This unusual mRNA editing apparently occurs before polyadenylation, probably in the nucleus.
Boström
K
K
Department of Physiology, Gladstone Foundation Laboratory for Cardiovascular Disease, University of California, San Francisco 94140-0608.
Lauer
S J
SJ
Poksay
K S
KS
Garcia
Z
Z
Taylor
J M
JM
Innerarity
T L
TL
D000230:Adenocarcinoma; D000818:Animals; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000235:genetics*; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D003110:Colonic Neoplasms; D004247:DNA / Q000235:genetics; D005784:Gene Amplification; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D008113:Liver Neoplasms; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D012313:RNA / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012313:RNA; D004247:DNA