title: Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells.
abstract: APOBEC1 is the catalytic subunit of the complex that edits ApolipoproteinB (ApoB) mRNA, which specifically deaminates cytidine 6666 to uracil in the human transcript. The editing leads to the generation of a stop codon, resulting in the synthesis of a truncated form of ApoB. We have developed a method to quantitatively assay ApoB RNA editing in live cells by using a double fluorescent mCherry-EGFP chimera containing a ∼ 300 bp fragment encompassing the region of ApoB subject to RNA editing. Coexpression of APOBEC1 together with this chimera causes specific RNA editing of the ApoB fragment. The insertion of a stop codon between the mCherry and EGFP thus induces the loss of EGFP fluorescence. Using this method we analyze the dynamics of APOBEC1-dependent RNA editing under various conditions. Namely we show the interplay of APOBEC1 with known interactors (ACF, hnRNP-C1, GRY-RBP) in cells that are RNA editing-proficient (HuH-7) or -deficient (HEK-293T), and the effects of restricted cellular localization of APOBEC1 on the efficiency of the editing. Furthermore, our approach is effective in assaying the induction of RNA editing in Caco-2, a cellular model physiologically capable of ApoB RNA editing.
authors: a Core Research Laboratory; Istituto Toscano Tumori ; Firenze , Italy.
authors: Francesco
authors: F
authors: Severi
authors: Silvestro G
authors: SG
authors: Conticello
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D017393:RNA Editing; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D014498:Uracil / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D014498:Uracil; D003596:Cytosine
keywords: ACF, APOBEC1 Complementation Factor; ADAR, Adenosine Deaminase, RNA-specific; ADAT, Adenosine Deaminase, tRNA-specific; AID/APOBECs; APOBEC1, Apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1; ApoB, Apolipoprotein B; EGFP, Enhanced Green Fluorescent Protein; FACS, Fluorescence activated cell sorting; FBS, Fetal bovine serum; GRY-RBP, Glycine-Arginine-Tyrosine-rich RNA-binding protein; RBM47, RNA binding motif protein 47; RNA editing; cds, coding sequence; cytosine deaminase; hnRNP-C1, heterogeneous nuclear ribonucleoprotein C1; lipid metabolism; mRNA; post-transcriptional modification