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

A Mehta, MT Kinter, NE Sherman… - Molecular and cellular …, 2000 - Taylor & Francis
A Mehta, MT Kinter, NE Sherman, DM Driscoll
Molecular and cellular biology, 2000Taylor & Francis
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) …
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.
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