Transient overexpression of exogenous APOBEC3A causes C-to-U RNA editing of thousands of genes

Abstract

APOBEC3A cytidine deaminase induces site-specific C-to-U RNA editing of hundreds of genes in monocytes exposed to hypoxia and/or interferons and in pro-inflammatory macrophages. To examine the impact of APOBEC3A overexpression, we transiently expressed APOBEC3A in HEK293T cell line and performed RNA sequencing. APOBEC3A overexpression induces C-to-U editing at more than 4,200 sites in transcripts of 3,078 genes resulting in protein recoding of 1,110 genes. We validate recoding RNA editing of genes associated with breast cancer, hematologic neoplasms, amyotrophic lateral sclerosis, Alzheimer disease and primary pulmonary hypertension. These results highlight the fundamental impact of APOBEC3A overexpression on human transcriptome by widespread RNA editing.

Keywords: Cytidine deaminase; RNA editing; RNA seq; disease genes; epitranscriptomics.

Figure 1.

Overexpression of A3A in 293T cells induces c.136 C >U SDHB RNA editing proportional to the increased amounts of A3A plasmid. (A) SDHB c.136 C > U editing levels in 293T cells are shown. The cells are transfected with control empty vector (500 ng) or pA3A at amounts indicated in a 12-well tissue culture plate. Mean and its standard error for n = 3 are shown. (B) Immunoblot showing A3A levels in whole-cell lysates (20 µg protein) of 293T cells transiently transfected with an empty vector control (EV) or increasing amounts of plasmid DNA construct for expression of A3A (pA3A). The A3A protein expression was detected by anti-DDK tag antibody. β-Actin is used as a loading control.

Figure 2.

Sanger validation of C > U RNA editing sites in primary cells identified by low-stringency RNA seq analysis of 293T/A3A cells. RNA editing at the highlighted Cs was validated in monocyte/enriched PBMCs (MEPs; n = 3 donors) treated with hypoxia and IFN1 (HI) for 24 h, in 7 of 19 tested genes. C > T(U) editing is characterized by emergence of a secondary T peak (red) accompanied by a reduction in height of C peak (blue). MEP-N shows lack of editing in a representative sequence chromatogram from control MEP cells maintained in normoxia without IFN1 treatment. Chromatograms for the remaining 12 RNA editing sites that could not be validated in MEPs are shown Fig. S2.

Figure 3.

Salient characteristics of C > (U)RNA editing by A3A in 293T cells (A) Mean and range of editing level at the 4,373 sites identified as targets for A3A-mediated editing are shown for the 3 A3A transfectant samples. The sites are ordered by the mean editing level. (B) Logo indicating sequence conservation and base frequency for sequences bearing the editing sites (at position 0). (C) Histogram of lengths in bases of inverted repeat sequences flanking the editing sites.