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

• Published in: Journal of molecular biology Vol. 431; no. 7; pp. 1506 - 1517
• Main Authors: Wolfe, Aaron D., Arnold, Don B., Chen, Xiaojiang S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 29.03.2019
• Subjects: Animals; APOBEC-1 Deaminase - chemistry; APOBEC-1 Deaminase - genetics; APOBEC-1 Deaminase - metabolism; APOBEC1; cell-based RNA editing assay; cofactor A1CF and RBM47; Gene Expression Regulation; HEK293 Cells; Humans; Mice; RNA Editing; RNA, Messenger - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; APOBEC1; RNA editing; RBM47; cofactor A1CF and RBM47; cell-based RNA editing assay; A1CF; NES; APO1
• DOI: 10.1016/j.jmb.2019.02.025
• ISSN: 0022-2836

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 versus 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. [Display omitted] •RNA editing is detectable by subcellular localization changes of fluorescence signals due to loss of nuclear export signal fused to GFP.•APO1 + RBM47 complex is more active than APO1 + A1CF in editing ApoB mRNA, the canonical substrate of APO1.•The N-terminal RBM47 domains necessary and sufficient for APO1 activity share high homology with A1CF, but the C-terminal domains not needed for RNA editing activity share little homology between the two proteins.•Editing can be observed for substrates other than APOB, and different editing efficiencies were observed depending on whether A1CF or RBM47 was used as the cofactor.•There are significant differences in substrate preferences between human APO1 + A1CF/RBM47 or mouse APO1 + A1CF/RBM47