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APOBEC3A Cytidine Deaminase Induces RNA Editing in Monocytes and Macrophages

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APOBEC3A Cytidine Deaminase Induces RNA Editing in Monocytes and Macrophages ARTICLE Received 8 Dec 2014 | Accepted 10 Mar 2015 | Published 21 Apr 2015 DOI: 10.1038/ncomms7881 OPEN APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages Shraddha Sharma1,*, Santosh K. Patnaik2,*, R. Thomas Taggart1, Eric D. Kannisto2, Sally M. Enriquez3, Paul Gollnick3 & Bora E. Baysal1 The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site- specific C4U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C4U RNA editing in mammals. 1 Department of Pathology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14203, USA. 2 Department of Thoracic Surgery, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14203, USA. 3 Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo, New York 14260, USA. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to B.E.B. (email: [email protected]). NATURE COMMUNICATIONS | 6:6881 | DOI: 10.1038/ncomms7881 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7881 NA editing is a co- or posttranscriptional process that alters Most importantly, we demonstrate that APOBEC3A, which transcript sequences without any change in the encoding belongs to the APOBEC3 family of CDAs, is an RNA-editing RDNA sequence1. Although various types of RNA editing enzyme. These findings significantly expand our understanding of have been observed in single-cell organisms to mammals, base C4U RNA editing and open new avenues of inquiry on the role modifications by deamination of adenine to inosine (A4I) or of APOBEC3 genes in viral and chronic diseases. cytidine to uracil (C4U) are the major types of RNA editing in higher eukaryotes. I and U are read as guanosine (G) and thymine Results (T), respectively, by the cellular machinery during messenger RNA translation and reverse transcription. RNA editing can SDHB RNA editing in IFN-treated MEPs and M1 macrophages. therefore alter amino acid sequences, thereby modifying and Similar to hypoxia, an IFN-rich microenvironment is another factor that monocytes are exposed to during inflammation. diversifying protein functions. Aberrant RNA editing is linked to 17 neuropsychiatric diseases such as epilepsy and schizophrenia, and IFNs also upregulate expression of APOBEC3 CDAs , candidate 4 chronic diseases such as cancer1. enzymes that may be responsible for the SDHB c.136C U RNA RNA-dependent ADAR1, ADAR2 and ADAR3 adenosine editing observed in monocytes. We therefore examined whether 4 deaminases, and APOBEC1 cytidine deaminase (CDA) are the IFNs induce SDHB c.136C U RNA editing. As shown in the left only known RNA-editing enzymes in mammals. RNA sequencing panel of Fig. 1a, treatment of MEPs with type 1 IFN (IFN1; studies suggest that A4I RNA editing affects hundreds of thousands of sites, although most of A4I RNA edits occur at a 70 Normoxia Hypoxia low level and in non-coding intronic and untranslated regions, 60 especially in the context of specific sequences such as Alu 60 2–4 elements .A4I editing of protein-coding RNA sequences at a 50 high level (420%) is rare and thought to occur predominantly in 50 the brain. Unlike A4I editing catalysed by adenosine 40 deaminases5, the prevalence and level of C4U RNA editing in 40 different types of cells and its enzymatic basis and regulation are 30 poorly understood. The activation-induced deaminase (AID), 30 apolipoprotein B-editing catalytic polypeptide-like (APOBEC) 136C>U editing (%) c. family and CDA proteins of mammals harbour the CDA motif 20 20 6 for hydrolytic deamination of C to U . The CDA enzyme is SDHB involved in the pyrimidine salvaging pathway. Although AID 10 10 causes C4U deamination of DNA, multiple studies have failed to 7 identify any RNA-editing activity for this protein . Humans have 0 0 – IFN1 γ 0 300 1,500 IFN1 (U ml–1) ten APOBEC genes (APOBEC1, 2, 3A–D, 3F–H and 4). APOBEC3 IFN proteins can deaminate cytidines in single-stranded (ss) DNA, 8 and although the APOBEC proteins bind RNA C4U 30 M0 deamination of RNA is known for only APOBEC1, with 14 M1 ) 9 2 M2 apolipoprotein B (APOB) mRNA as its physiological target . 12 25 C4U RNA editing alters hundreds of cytidines in chloroplasts and mitochondria of flowering plants, but the underlying 10 10 20 deaminating enzymes are unknown . 8 We have previously observed C4U editing of cytidine at c.136 6 (NCBI reference sequence NM_003000), which generates a 15 nonsense codon (R46X), in B6% of transcripts of the succinate 4 dehydrogenase B (SDHB) gene in normal peripheral blood c. 136C>U editing (%) c. 10 mononuclear cells (PBMCs) of humans11. SDHB encodes the 2 iron-sulfur subunit of mitochondrial respiratory complex II, 0 SDHB 5 which also participates in oxygen sensing and response12–14. M0 (log Expression change versus Mutations in SDH genes are associated with both hereditary and –2 non-hereditary paraganglioma and pheochromocytoma, renal 0 CCL2 CCL19 FCER2 MRC1 carcinoma and gastrointestinal stromal tumours15. More recently, we found that hypoxia (1% O2) enhances the C4U editing of Figure 1 | SDHB c.136C4U RNA editing in IFN-treated MEPs and M1 SDHB RNA at c.136 in monocytes, with an editing level of B18% macrophages (a) Mean and its s.e. (n ¼ 3) are shown on left for editing observed for monocyte-enriched PBMCs (MEPs) after 48 h of levels in MEPs optionally treated with IFN1 (600 U ml À 1), IFNg 16 À 1 hypoxia . Monocytes infiltrate tumours, atheromatous plaques (200 U ml ) and hypoxia (1% O2) for 24 h. The additive induction of SDHB and sites of infection and inflammation, which are characterized c.136C4U RNA editing by the IFNs and hypoxia is also depicted on right. by micro-environmental hypoxia. C4U RNA editing of SDHB Matched MEPs of seven individuals were cultured under normoxia or may therefore represent a hypoxia-adaptive mechanism that may hypoxia with 0, 300 or 1,500 U ml À 1 IFN1 for 24 h. Mean and its s.e. (n ¼ 7) have implications for the pathogenesis of chronic inflammatory for editing levels in the cells are shown. Editing level in cells treated with diseases. both hypoxia and IFN1 was higher than in cells treated with only hypoxia or To identify additional C4U RNA editing events in monocytes IFN1 (Wilcoxon test Po0.02, for both concentrations of IFN1). (b) M1 and and monocyte-derived macrophages (MEPs), we analyse their M2 macrophages were generated from unpolarized M0 macrophages whole transcriptome RNA sequences. We show that transcripts of derived from CD14 þ monocytes isolated from peripheral blood of three hundreds of genes including those implicated in viral pathogen- individuals. Mean and range (n ¼ 3) of expression of genes for markers of esis and Alzheimer’s disease are targets of editing in monocytes M1 and M2 polarization and SDHB c.136C4U editing levels in the cells are and macrophages. Such editing is regulated by oxygen, depicted. Gene expression was quantified by RT–PCR and normalized to interferons (IFNs) and also during macrophage polarization. that of ACTB. 2 NATURE COMMUNICATIONS | 6:6881 | DOI: 10.1038/ncomms7881 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7881 ARTICLE 600 U ml À 1) or IFNg (200 U ml À 1) for 24 h induced SDHB and 6.6% of the sites, respectively, whereas these two types of c.136C4U RNA editing in MEPs, both in normoxia and hypoxia editing respectively occurred at 10.6% and 86.5% of the sites in under 1% O2 (Mann–Whitney U-test Po0.01, comparing macrophages. A4G editing occurred at an overwhelming untreated and IFN-treated samples). The editing level in majority of the sites in MEPs, but only 1.0% of the A4G sites normoxic or hypoxic MEPs was increased B6-fold by IFN1 were in coding exons, causing 18 non-synonymous and 12 and B3-fold by IFNg, suggesting that the induction of RNA synonymous codon changes (Fig. 2c and Supplementary Table 5). editing with IFN1 was higher than with IFNg (Wilcoxon rank This is consistent with the known targeting of A4G RNA editing sum test Po0.03, comparing samples regardless of hypoxia to non-coding sequences4. On the other hand, 61.7% of the total treatment). An additive effect of IFNs and hypoxia on SDHB 211 C4U sites (in 199 genes) were in coding exons, causing 55 c.136C4U RNA editing was observed and this was confirmed in non-synonymous and 73 synonymous codon changes. C4U an independent experiment in which matched MEPs of seven editing accounted for 73.3% of all non-synonymous editing individuals were cultured under normoxia or hypoxia with 0, 300 upregulated by hypoxia in MEPs. In macrophages, 77.9% of the or 1,500 U ml À 1 IFN1 for 24 h. Editing level in cells treated with total 122 C4U sites (in 116 genes) were in coding exons, causing both hypoxia and IFN1 was higher than in cells treated with only 27 non-synonymous and 66 synonymous codon changes.
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