Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA

Abstract

The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.

FIG. 1

Protein sequence of ACF. (A) The deduced amino acid sequence of the ACF cDNA is shown. The peptide sequences obtained from mass spectrometric analysis are underlined. (B) A diagram of the three RRM motifs in ACF and the alignment of residues 58 to 123, 138 to 208, and 233 to 293 with the consensus RRM motif (4) containing the conserved RNP2 hexamer and RNP1 octamer sequences.

FIG. 2

Expression of ACF mRNA. (A) A human tissue Northern blot (Clontech) was probed with ³²P-labeled ACF cDNA insert. The positions of RNA molecular weight markers are shown on the left. (B) Multiple tissue cDNA panels (Clontech) were analyzed for the ACF and GAPDH cDNAs by PCR.

FIG. 3

ACF and apobec-1 edit apo-B RNA in vitro. (A) Purified recombinant His₆-ACF and His₆–apobec-1 were resolved by SDS-PAGE and stained with Coomassie blue (ACF) or silver (apobec-1). (B) Purified His₆–apobec-1 (∼0.5 ng) and His₆-ACF (∼0.6 ng) were added to in vitro editing assays containing wild-type apo-B RNA, the triple mutant RNA with three mutations in the mooring sequence, or the point mutant RNA (U⁶⁶⁷⁸→G). After incubation at 30°C for 2 h, the reactions were analyzed by a poisoned primer extension assay (28). The positions of the products from the edited (UAA) and unedited (CAA) RNAs are indicated. (C) In vitro editing assays were performed with His₆–apobec-1 and His₆-ACF (∼1.5 ng) or with rat liver extracts (60 μg). After incubation at 30°C for the indicated times, the reactions were analyzed as described above.

FIG. 4

ACF UV cross-links to apo-B RNA. Purified His₆-ACF (0.6 ng) was used in UV cross-linking experiments with ³²P-labeled wild-type apo-B RNA. Competition experiments were performed with a 5- to 50-fold molar excess of the unlabeled wild-type apo-B RNA, the triple mutant RNA with three mutations in the mooring sequence, or the point mutant RNA (U⁶⁶⁷⁸→G) as indicated. Reactions were analyzed by SDS–8% PAGE and autoradiography.

FIG. 5

ACF coimmunoprecipitates with apobec-1 in transfected cells. (A) Extracts from Cos-7 cells were transiently transfected with vector DNA or plasmids encoding the ACF and apobec-1 cDNAs as indicated. After 48 h, cell extracts were analyzed by Western blotting using anti-ACF(4-18) or anti-apobec-1 antibodies. (B) The extracts from transfected Cos-7 cells were immunoprecipitated with the anti-ACF(4-18) antibody coupled to protein A-Sepharose. After extensive washes, the complexes were resolved on SDS–12% PAGE, transferred to PVDF membranes, and analyzed by Western blotting using the anti-ACF(4-18) or anti-apobec-1 antibodies as indicated.

FIG. 6

Immunodepletion of editing activity from rat liver extracts. (A) Rat liver extracts were incubated with anti-ACF(4-18) or anti-ACF(408-422) antibodies or their respective preimmune sera (PI) as indicated. The immune complexes were removed by protein A-agarose and the supernatants were analyzed in an in vitro editing assay. (B) The protein A-Sepharose beads containing the immune complexes from above were extensively washed. Aliquots of the beads (5 to 15 μl) were incubated with synthetic apo-B RNA in an in vitro editing assay.

FIG. 7

In vivo association of ACF with apo-B mRNA. Nuclear extracts from McArdle 7777 cells were immunoprecipitated with the anti-ACF(4-18) antibody or preimmune serum as described in the legend to Fig. 6. RNAs extracted from nuclear extracts or the immune complexes were analyzed by reverse transcriptase PCR using gene-specific primers for GAPDH or apo-B. Reverse transcriptase (RTase) was included in the cDNA reaction mixture as indicated. The PCR products were analyzed by electrophoresis on a 1.2% agarose gel. The positions of the GAPDH and apo-B products are indicated.