(Received for publication, January 31, 1996; and in revised form, February 27, 1996)
From the
A site-specific C to U editing reaction modifies nuclear
apolipoprotein B100 (apoB100) mRNA, producing apolipoprotein B48 in the
mammalian small intestine. This reaction is mediated by a
multicomponent enzyme complex, which contains a catalytic subunit,
Apobec-1. We have used gene targeting to disrupt mouse apobec-1 in order to establish its requisite importance in apoB mRNA
editing and also, in view of its widespread tissue distribution in
rodents, as a preliminary indication of other potential roles. Both
heterozygous (apobec-1) and
homozygous (apobec-1
) gene-targeted
mice appear healthy and fertile with no alterations in serum
cholesterol or triglyceride concentrations. The apobec-1
mice demonstrated reduced
levels of hepatic apoB mRNA editing. By contrast, levels of small
intestinal apoB mRNA editing were indistinguishable in wild-type and apobec-1
animals, suggesting that
Apobec-1 is expressed in limited quantities in the liver but not in the
small intestine. The apobec-1
mice
lacked detectable levels of Apobec-1 mRNA, expressed only unedited apoB
mRNA in all tissues, and contained no apoB48 in their serum,
demonstrating that there is no functional duplication of this gene.
A common structural gene encodes two distinct forms of mammalian
apolipoprotein B (apoB), ()in proportions that are
determined by tissue-specific RNA
editing(1, 2, 3) . ApoB mRNA editing occurs
in the small intestine, where a site-specific cytidine deamination
reaction at position 6666 in the nuclear transcript creates a UAA stop
codon and generates a truncated protein,
apoB48(2, 3) . This same process is operative in the
liver of some species, such as the rat and
mouse(4, 5, 6, 7, 8) .
Human liver, on the other hand, contains only unedited apoB mRNA and
secretes the full-length form, apoB100(1, 9) . ApoB100
contains the functional domains required for binding to the low density
lipoprotein receptor as well as the cysteine residue required for its
association with apolipoprotein(a) (1, 10, 11) . By corollary, the absence of
these domains from apoB48 has major functional consequences for
lipoprotein catabolism and may influence the atherogenic potential of
the apoB-containing lipoproteins (reviewed in (1) ).
ApoB
mRNA editing is mediated by a multicomponent enzyme complex that
includes a 27-kDa component, Apobec-1, as well as other as yet
uncharacterized factors that are required for the functional integrity
of the complex(12, 13, 14) . The primary
structure of Apobec-1 reveals homology to other cytidine and
deoxycytidine deaminases, particularly within a conserved motif
His-(X)-Cys-X-X-Cys (15, 16) . Furthermore, Apobec-1 demonstrates
zinc-dependent cytidine deaminase activity (15) against both
the cytidine at position 6666 of mammalian apoB mRNA as well as a
simple monomeric cytidine substrate(17) . The biological
significance of this latter activity and its role, if any, in the
regulation of cellular nucleoside metabolism are currently unknown.
In attempting to ascribe an integrated, biological role for Apobec-1, it was difficult to make a priori predictions, since recent reports suggest a range of potential functions. For instance, in addition to its activity as an apoB RNA-directed cytidine deaminase, Apobec-1 is an RNA binding protein, with broad specificity for AU-rich templates(16, 18) . Other features suggest the possibility of a biological role for Apobec-1 distant from its function in apoB mRNA editing. These include the observation that Apobec-1 is widely expressed and developmentally regulated in both rat and mouse tissues, including many sites such as the kidney, lung, and spleen, which contain trivial or undetectable amounts of apoB mRNA(13, 19, 20) . Thus, although restricted to the gastrointestinal tract in humans and rabbits(21, 22, 23, 24) , the broad tissue distribution of Apobec-1 in rats and mice, taken together with the RNA binding and cytidine deaminase activity alluded to above, implies the possibility that potential functions exist for this protein beyond its role in apoB mRNA editing. Consistent with this suggestion is the recent report that transgenic mice and rabbits that overexpress Apobec-1 in the liver tend to develop hepatic dysplasia and hepatocellular carcinoma(25) . This study suggested that it is possible that other mRNA substrates, in addition to apoB, might undergo editing(25) .
In order to determine the potential biological
functions of Apobec-1 in vivo, we have used gene targeting to
create mice that are homozygous for an apobec-1 gene knockout.
We report that homozygous apobec-1 knockout mice (apobec-1) appear healthy and
fertile and have no obvious abnormalities other than the absence of
plasma apoB48 and undetectable apoB mRNA editing. Heterozygous (apobec-1
) mice manifested reduced
levels of apoB mRNA editing in the liver; however, apoB mRNA editing
was indistinguishable in the small intestine of apobec-1
and wild-type animals. These
findings suggest that Apobec-1 is normally present in excess in the
mouse small intestine but at limiting concentrations in the liver.
Figure 1:
Targeting strategy for apobec-1 gene knockout. A, schematic map of the wild-type (WT) allele showing 8 exons. A replacement vector was
constructed using a 4.5-kb BamHI fragment and a 2.5-kb HindIII fragment, which were ligated into pNTK (see
``Materials and Methods''). The disrupted allele lacks exon 6
and contains new PvuII and NcoI sites. B,
Southern blot of DNA from representative G418-resistant and
(1-(2`-deoxy-2`-fluoro--D-arabinofuranosyl)-5`-iodouracil
resistant ES clones, digested with either NcoI (N) or PvuII (P) and probed with a 0.8-kb HindIII/SalI fragment (hatched bar, panel A). Targeted clones were verified by Southern blotting
using a neo probe (data not shown). C, Southern blot
of tail DNA from three wild-type (+/+), one apobec-1
(+/-), and two apobec-1
animals (-/-),
digested with PvuII and probed as in panel B. B, BamHI; H, HindIII; P, PvuII; N, NcoI; TK, thymidine
kinase cassette; Neo, neomycin resistance
cassette.
Figure 2:
Screening strategy for apobec-1 gene knockout. Two independent PCR reactions were used. One
reaction was used to determine the absence of exon 6 of apobec-1, using primers P1 and P2. A second reaction was used
to demonstrate the presence of the neo cassette, using primers
P3 and P4. A, absence of exon 6 from genomic DNA of apobec-1 mouse. M,
X
markers. WT, wild type. B, presence of neo cassette in both apobec-1
and apobec-1
mice. M,
HindIII markers. C, RNase protection assay on total
hepatic and small intestinal RNA (50 µg each), using a 446-nt SalI/KpnI probe to demonstrate the reduction in
Apobec-1 mRNA abundance in apobec-1
(+/-) mice (protected fragment, 422 nt) and its
absence in apobec-1
(-/-) mice. The abundance of the Apobec-1 mRNA was
normalized using a 333-nt mouse
-actin probe, which yielded a
247-nt protected fragment.
Figure 3: Immunodetection of apoB in plasma. 1-µl aliquots of plasma from mice of the representative genotype were electrophoresed through denaturing 4% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The blots were probed with a rabbit anti-rat apoB antiserum, and the reaction was developed using enhanced chemiluminescence. The migrations of apoB100 and apoB48 are shown.
Figure 4: ApoB mRNA editing in apobec-1-targeted mice. Total RNA was extracted from the indicated tissues and subjected to DNase I treatment prior to reverse transcription-PCR amplification of apoB cDNA. To validate the absence of contaminating genomic DNA, samples were run with (+) or without(-) reverse transcription. The PCR products were purified and used for primer extension analysis. A, small intestine; B, liver; C, kidney. Genotypes are illustrated for each tissue. The positions of CAA and TAA bands, corresponding to the unedited and edited apoB cDNA, respectively, are illustrated.
The availability of apobec-1 mice will allow us to
examine issues relating to the organizational state and function of the
complementation factors of the apoB mRNA editing enzyme, specifically
in tissue extracts that now express no detectable quantities of the
catalytic subunit. It will be informative, for example, to examine
intestinal and hepatic extracts prepared from these apobec-1
mice for the presence of
the RNA binding activities, which correspond to p44 and
p60(28, 29) , in order to determine whether these
functional attributes are retained in the absence of Apobec-1.
In
addition, it will be of importance to determine whether apobec-1 mice are susceptible to
atherosclerosis particularly in view of the fact that their serum
contains exclusively apoB100. In this regard, their phenotype is
similar to that of mice which have been recently generated through
targeting of the edited codon of the endogenous apoB gene,
such that they express only murine apoB100. (
)The outcome of
such studies, particularly with respect to the need for a high fat,
cholesterol-enriched diet in order to induce
atherosclerosis(30) , will be of intense interest. These and
other issues will be the focus of future reports.