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

Author:
Wolfe, Aaron D., Arnold, Don B., Chen, Xiaojiang S.
Source:
Journal of molecular biology 2019 v.431 no.7 pp. 1506-1517
ISSN:
0022-2836
Subject:
RNA, RNA editing, cell lines, cytosine deaminase, fluorescence, gene expression, genes, humans, lipid metabolism, mice
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 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.
Agid:
6328087