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Apolipoprotein (apo) B mRNA editing consists of a C
Volume 270,
Number 22,
Issue of June 2, pp. 13042-13056, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
STRUCTURE AND EVOLUTION OF Apobec1 AND RELATED
NUCLEOSIDE/NUCLEOTIDE DEAMINASES
U
conversion involving the first base of the codon CAA, encoding Gln
2153, to UAA, a stop codon. Editing occurs in the intestine only in
most mammals, and in both the liver and intestine in a few mammalian
species including mouse. We have cloned the cDNA for the mouse apoB
mRNA editing protein, apobec1. Expression of mouse apobec1 cDNA in
HepG2 cells results in the editing of the intracellular apoB mRNA. The
cDNA predicts a 229-amino acid protein showing 92, 66, and 70% identity
to the rat, rabbit, and human proteins, respectively. Based on the
estimated values of divergence of apobec1 sequences in terms of the
numbers of synonymous and non-synonymous substitutions per site, we
found that apobec1 is a fairly rapidly evolving protein. Sequence
comparison among mammalian apobec1 sequences has permitted the
identification of seven conserved regions that may be functionally
important for editing activity. We present a phylogenetic tree relating
apobec1 sequences to double-stranded RNA adenosine deaminase and other
nucleotide/nucleoside deaminases. Northern blot analysis indicates that
apobec1 mRNA exists in two different sizes, a
2.2-kilobase (kb)
form in small intestine and a
2.4-kb form in liver, spleen,
kidney, lung, muscle, and heart. To study the molecular basis for the
different sized apobec1 mRNAs, we cloned the apobec1 gene and
characterized its exon-intron organization together with the sequences
expressed in the hepatic and intestinal mRNA. The mouse apobec1 gene
contains 8 exons and spans
25 kb, and is located in chromosome 6.
The major hepatic mRNA contains all 8 exons, whereas the major small
intestinal mRNA misses the first 3 exons and its transcription is
initiated in exon 4. The intestinal mRNA also contains at its 5` end a
unique 102-nucleotide piece that is absent in the liver mRNA. We also
identified two alternatively spliced hepatic apobec1 mRNAs with
different acceptor sites in exon 4. Transient expression studies using
promoter-reporter gene constructs in HeLa, Hepa, and Caco-2 cells
indicate that the 5`-flanking sequences of the liver mRNA (i.e. upstream of exon 1) have predominantly hepatic promoter activity
and the 5`-flanking sequences of the major small intestine mRNA
(i.e. upstream of exon 4) have preferential intestinal
promoter activity. We conclude that alternative mRNA splicing and
differential promoter utilization determine the tissue-specific
expression of the apobec1 gene in mice. Our observations provide
information on unique aspects of the molecular basis of the
tissue-specific expression of apobec1 mRNA and the evolutionary basis
of RNA editing.
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