Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells

Abstract

RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed that APOBEC1 may be involved in editing other potential RNA targets in a tissue specific manner, and another protein, RBM47, appears to instead be the main cofactor of APOBEC1 for editing APOB RNA. In this report, by expressing APOBEC1 with either A1CF or RBM47 from human or mouse in an HEK293T cell line with no intrinsic APOBEC1/A1CF/ RBM47 expression, we have compared direct RNA editing activity on several known cellular target RNAs. By using a sensitive cell-based fluorescence assay that enables comparative quantification of RNA editing through subcellular localization changes of eGFP, the two APOBEC1 cofactors, A1CF and RBM47, showed clear differences for editing activity on APOB and several other tested RNAs, and clear differences were observed when mouse vs. human genes were tested. In addition, we have determined the minimal domain requirement of RBM47 needed for activity. These results provide useful functional characterization of RBM47 and direct biochemical evidence for the differential editing selectivity on a number of RNA targets.

The structure of APOBEC1 and insights into its RNA and DNA substrate selectivity

“…To validate the RNA deamination activity seen with the in vitro poisoned primer extension and further characterize the RNA editing activity of APO1 mutations in a cellular environment, we used an in-cell fluorescence assay to detect and quantify RNA editing activity as previously described ( 33 ). This fluorescence assay can detect editing of a fraction of mRNA molecules of a reporter protein by visualizing/quantifying the fluorescent shift into the nucleus from the cytosol.…”

“…Editing of the targeted C to U on the mRNA creates an early stop codon before the NES, resulting in a quantifiable shift of eGFP fluorescence from the cytosol to the nucleus (Figure 6A ), which can be used as a sensitive reporter for RNA editing by APO1/cofactor expressed from an editor construct in a cellular environment. The RNA region used here for this cell-based assay is a 27-nt segment of human APOB RNA that has been shown to be specifically edited by APO1 when paired with A1CF (or RBM47) in previous reports ( 33 , 45 , 78 ). The editor construct expresses APO1, A1CF and mCherry from the same open reading frame of a single mRNA, but a 2A peptide ( 79 , 80 ) is inserted between each protein to allow the generation of individual proteins during translation ( Supplementary Figure S6A ).…”

“…An HRV protease site was inserted via mutagenesis into the linker region between the MBP and A1CF domains in order to allow for removal of the fusion protein, and a stop codon at the 392 positions of A1CF was later added to produce A1CF protein containing residues 1–391 experiments within this report. The reporter and editor constructs were derived from a previous study ( 33 ). All cloning was done into chemically competent Stellar (Takara Biosciences), TOP10 or DH5α cells.…”

“…Fluorescence localization assays were performed as previously described ( 33 ). Briefly, the reporter and editor constructs were co-transfected into HEK 293T cells using X-TREME gene 9 transfection reagent (Sigma) using a 10:1 mass ratio excess of editor to reporter plasmid.…”

“…The classical A1CF (APOBEC1 complementation factor) ( 30 ) and the newly discovered RBM47 (RNA-binding protein 47) ( 31 ) are the two cofactors identified so far. A1CF and RBM47 can individually confer editing activity by APO1 on known RNA substrates and are important for lipid uptake and regulating LDL levels ( 32 , 33 ). In addition, A1CF and RBM47 are essential genes ( 31 , 32 , 34 , 35 ), and play a role in RNA processing (splicing and editing), liver development and kidney function, and cancer ( 34 , 36–38 ).…”