Overexpression of human APOBEC3B in human HEK-293T cells
To identify transcriptome-wide effects of human APOBEC3B, human HEK-293T cells were transiently transfected with a either an empty vector control construct (pcDNA3.1+) or an expression construct for human APOBEC3B ORF cDNA. Comparison of transcriptome sequences identified hitherto unknown RNA editing activity of human APOBEC3B, a novel finding that was presented at the annual meeting of RNA Society in July 2016 in Kyoto, Japan as follows: TITLE: APOBEC3B is a new RNA editing enzyme. AUTHORS: Santosh Patnaik and Eric Kannisto. ABSTRACT: The prevalence of C>U type of RNA editing and its enzymatic basis, biological consequence, and regulation are poorly understood. For almost 25 years, APOBEC1 has been the only protein known to cause C>U editing of mRNAs in mammals. We recently observed site-specific C>U editing of scores of mRNAs in human macrophages and showed that such editing is physiologically regulated, occurring in cells in pro-inflammatory M1 but not anti-inflammatory M2 polarization state. We analyzed the basis of this and discovered APOBEC3A, a member of the AID/APOBEC family of cytidine deaminases, to be a new C>U RNA editing enzyme. APOBEC3B is a homolog of APOBEC3A. It is expressed in embryonic stem cells, immune cells and some epithelial tissues, and is well-known for restricting retrotransposons and viruses. A deletion allele of APOBEC3B that is seen in certain populations increases risk for developing cancer. APOBEC3B can deaminate DNA and is thought to be a genome mutator.  To assess if APOBEC3B can edit RNA, like APOBEC3A, we exogenously expressed the protein in APOBEC3B-deficient 293T human embryonic kidney cells. Whole exome and transcriptome sequences of  these cells were compared to those of control 293T transfectants. We thus identified 211 APOBEC3B-induced C>U RNA sequence changes that were present at frequencies of 4% to 50% for a total of 206 genes and occurred without a corresponding DNA sequence variation. Almost half (102) of the 211 RNA edits cause nonsense or non-synonymous codon changes resulting in changes to proteins encoded by the APOBECB target RNAs. RNA editing for 18 genes was examined and validated in another set of 293T transfectants by Sanger sequencing of cDNA and genomic DNA amplicons. In vitro, APOBEC3B protein deaminated cytidines of a target RNA to cause site-specific C>U editing. Studies of single-residue mutants of APOBEC3B showed that its C-terminal Z1 but not N-terminal Z2 deaminase domain is required for RNA editing.  We have thus identified APOBEC3B as a novel RNA editing enzyme. The hitherto unknown RNA editing capability of APOBEC3B may play a role for its biological function as a restrictor of retrotransposons and viruses, and may be of relevance to development of cancer.