RNA editing of apolipoprotein B mRNA. Sequence specificity determined by in vitro coupled transcription editing.
Apolipoprotein (apo) B-48 mRNA is produced by in vivo RNA editing which involves a C----U conversion of the first base of the codon CAA for Gln-2153, changing it to UAA, an in-frame stop codon. We have reproduced the editing reaction in vitro using nuclear extracts. Efficient RNA editing was demonstrated by using apoB mRNA segments as substrate or in a coupled transcription-editing reaction using apoB minigenes as template. ApoB minigenes were constructed by ligating the adenovirus major late promoter to a fragment of apoB-100 DNA containing the editing site and used for the transcription-editing reaction. We defined the sequence specificity of the editing reaction using site-specific single and multiple base mutants constructed by the polymerase chain reaction. Among 22 different mutant apoB-100 minigene constructs containing mutations in the bases immediately flanking the edited C-6666, 20 were edited in the coupled transcription-editing reaction. The results suggest a relatively lax sequence specificity for apoB mRNA editing. Our observation may have important implications for apoB-48 biogenesis as well as for the editing process as a general biologic regulatory mechanism.
Chen
S H
SH
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Li
X X
XX
Liao
W S
WS
Wu
J H
JH
Chan
L
L
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003062:Codon / Q000235:genetics; D005796:Genes; D014644:Genetic Variation; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic
D001055:Apolipoproteins B; D003062:Codon; D015345:Oligonucleotide Probes; D012333:RNA, Messenger