Alternative Splicing of APOBEC3D Generates Functional Diversity and Its Role As a DNA Mutator
Authors: Takei Hisashi , Fujii Masanori , Nakayama Sohei , Kobayashi Ikei , Shindo Keisuke , Shirakawa Kotaro , Takaori-Kondo Akifumi , Kobayashi Susumu 
Published: October 2019

Abstract
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) protein family consists of 11 members: APOBEC1, APOBEC2, seven APOBEC3s (A3s) (A/B/C/D/F/G/H), APOBEC4, and the activation-induced deaminase (AID). APOBEC1, A3s, and AID have cytidine deaminase activity and induce a cytidine (C) to thymidine (T) transition. The main function of A3s is to trigger an innate immune response to viral infection such as human immunodeficiency virus-1. Recently, it was reported that several APOBEC family proteins can induce somatic mutations into genomic DNA and thus promote cancer development. For example, AID-mediated somatic mutations contribute to B-cell lymphoma and A3B can be an enzymatic source in solid tumors such as breast cancer. However, little is known about other A3 member proteins.
To determine the expression of A3 in hematopoietic cells, we performed quantitative PCR in leukemia cell lines. A3B, A3C, A3D, A3F and A3G were detected in all cell lines, but not A3A or A3H. Next, we analyzed published The Cancer Genome Atlas data from patients with acute myeloid leukemia to find somatic alterations in A3 genes using cBioPortal (http://www.cbioportal.org/). We found that each A3 was upregulated at 4-7 % of all samples and the total frequency of 23 %. Interestingly, overall survival of patients with A3upregulation was lower than those without upregulation, suggesting an important role of A3s in pathogenesis of leukemia. As it has been shown that A3A, A3B, and A3D show capacity to inflict DNA damage, we decided to investigate whether A3D can induce mutations in foreign and genomic DNA.
During our attempt to generate A3D expression constructs, we detected multiple bands in leukemia and lung cancer cell lines. Based on our analysis and previous reports, we confirmed that there are at least 7 transcript variants (v1 to 7). Because A3D v3, v4 and v5 lack cytidine deaminase domains, we decided to examine A3Dv1, v2, v6 and v7.
To determine whether A3D variants have foreign DNA editing activity, we performed differential DNA denaturation (3D)-PCR in HEK293T cells co-transfected with expression vectors for EGFP, UNG inhibitor, and pCAG-GS containing A3Dv1, v2, v6, v7, A3B or empty control. Total DNA was isolated 4 days after transfection. PCR products were detected at lower denaturation temperature (Td) in cells expressing A3B and all A3D variants vectors compared to control. Sequences of PCR products at lowest Td revealed that mutation frequencies in EGFP gene were 6.1-10.5 per 103 bps in cells transfected with all A3D variants and 18.7 per 103bps in cells transfected with A3B. Of note, 90 % of mutations were C/G to T/A transition in cells transfected with A3B, but in cells transfected with A3D these were 30-60 %.
Next we performed 3D-PCR in HEK293T cells retrovirally infected with MigR1-IRES-EGFP containing A3Dv1, v2, v6, v7, A3B or empty to determine genomic DNA editing activity. Total DNA was isolated 4 weeks after infection. At lower Td, PCR products were detected only in cells expressing A3Dv1 and A3B. PCR products in cells expressing A3Dv2, v6, v7 were detected only at same Td as control. Sequences of the PCR products at lowest Td revealed that the mutation frequency in the EGFP gene was 22.2 per 103 bps in cells expressing A3Dv1, and 56.9 per 103 bps in cells expressing A3B. However mutation frequencies in cells expressing A3Dv6 or v7 were lower than 2 per 103bps and no mutations were detected in cells expressing A3Dv2. All of these mutations were C/G to T/A transition. Interestingly, we found 4 clones with deletion in cells expressing A3Dv1, v6 and A3B. In addition, all clones had several bases of micro homologous sequences in broken ends before joining. These results suggested that double strand breaks in genomic DNA were repaired by microhomology-mediated end joining.
These DNA editing assays showed that all A3D variants had ability to induce mutations in foreign DNA, and A3Dv1 was major isoform which had ability to induce mutations in genomic DNA.
Taken together, our experiments showed that A3D can be a DNA mutator and alternative splicing generates functional diversity of A3D. These findings suggest that A3D may contribute to development of hematopoietic malignancies.

Disclosures
Takaori-Kondo: Mochida Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding; Takeda Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Kyowa Kirin: Research Funding; Pfizer: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.