Hypoxia-inducible C-to-U coding RNA editing downregulatesSDHBin monocytes

Abstract

Background. RNA editing is a post-transcriptional regulatory mechanism that can alter the coding sequences of certain genes in response to physiological demands. We previously identified C-to-U RNA editing (C136U, R46X) which inactivates a small fraction of succinate dehydrogenase (SDH; mitochondrial complex II) subunit B gene (SDHB) mRNAs in normal steady-state peripheral blood mononuclear cells (PBMCs). SDH is a heterotetrameric tumor suppressor complex which when mutated causes paraganglioma tumors that are characterized by constitutive activation of hypoxia inducible pathways. Here, we studied regulation, extent and cell type origin of SDHB RNA editing.Methods. We used short-term cultured PBMCs obtained from random healthy platelet donors, performed monocyte enrichment by cold aggregation, employed a novel allele-specific quantitative PCR method, flow cytometry, immunologic cell separation, gene expression microarray, database analysis and high-throughput RNA sequencing.Results. While the editing rate is low in uncultured monocyte-enriched PBMCs (average rate 2.0%, range 0.4%–6.3%, n = 42), it is markedly upregulated upon exposure to 1% oxygen tension (average rate 18.2%, range 2.8%–49.4%, n = 14) and during normoxic macrophage differentiation in the presence of serum (average rate 10.1%, range 2.7%–18.8%, n = 17). The normoxic induction of SDHB RNA editing was associated with the development of dense adherent aggregates of monocytes in culture. CD14-positive monocyte isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures (n = 5) and 1.68-fold in hypoxic cultures (n = 4). CD14-negative lymphocytes showed no evidence of SDHB editing. The SDHB genomic DNA remained wild-type during increased RNA editing. Microarray analysis showed expression changes in wound healing and immune response pathway genes as the editing rate increased in normoxic cultures. High-throughput sequencing of SDHB and SDHD transcripts confirmed the induction of C136U RNA editing in normoxic cultures but showed no additional verifiable coding edits. Analysis of SDHB RNA sequence data from 16 normal human tissues from the Illumina Body Map and from 45 samples representing 23 different cell types from the ENCODE projects confirmed the occurrence of site-specific C136U editing in whole blood (1.7%) and two primary CD14+ monocyte samples (1.9% and 2.6%). In contrast, the other cell types showed an average of 0.2% and 0.1% C136U editing rates in the two databases, respectively.Conclusions. These findings demonstrate that C-to-U coding RNA editing of certain genes is dynamically induced by physiologically relevant environmental factors and suggest that epigenetic downregulation of SDHB by site-specific RNA editing plays a role in hypoxia adaptation in monocytes.

Oncometabolites in cancer aggressiveness and tumour repopulation

“…The most frequently mutated SDH genes are SDHB and SDHD , with only a few mutations detected in SDHC , whereas SDHA or SDHAF genes are rarely mutated (Kim et al , ; Tretter, Patocs, & Chinopoulos, ). Besides the mutational status of SDH subunits, it is emerging that SDH expression/activity might also be regulated: ( i ) at a transcriptional or post‐transcriptional level, by epigenetic suppression of SDH subunit expression (Baysal, ; Richter et al , ), by RNA non‐sense mutation due to C‐to‐U RNA editing that has been demonstrated to reduce the SDHB functional transcript dynamically in monocytes (Baysal et al , ), and by the expression of microRNAs (miRNAs) targeting SDH messenger RNAs (mRNAs) (Eichner et al , ; Tsang et al , ; Lee et al , ); and ( ii ) at a post‐translational level, by the inhibition of SDH activity, for example through the binding of the chaperone tumour necrosis factor receptor‐associated protein 1 (TRAP‐1) (Sciacovelli et al , ) or through the competitive inhibition of the metabolite itaconate in activated macrophages (Lampropoulou et al , ), or by the regulation of SDH phosphorylation through protein tyrosine phosphatase mitochondrial 1 (PTPMT1) (Nath et al , ), feline Gardner‐Rasheed sarcoma viral oncogene homolog (Fgr) tyrosine kinase (Salvi et al , ), or avian sarcoma (Schmidt‐Ruppin A‐2) viral oncogene homolog (c‐Src) tyrosine kinase (Ogura et al , ).…”

Succinate Supplement Elicited “Pseudohypoxia” Condition to Promote Proliferation, Migration, and Osteogenesis of Periodontal Ligament Cells

“…The supplement of succinate is similar to succinate metabolism in the ischemia-reperfusion model, and elevated succinate may upregulate SDH transcription by substrateenzyme interaction [44], leading to enhanced SDH transcription as Figure 4(d). As shown in Figure 4(a), intracellular succinate levels peaked at 6 h; such high concentration of intracellular succinate may significantly affect its protein expression and activity by several possible mechanisms: succinate may stabilize HIF-1α to mimic hypoxic state, which triggers hypoxia-inducible C-to-U coding RNA editing [45], and miRNA-mediated epigenetic regulation may also contribute to transcription modification [46]; in addition, extensive ROS generation by reverse electron transport (RET) at mitochondrial complex I during oxidation of succinate may critically affect the availability of reduced NAD + and altered activity of SIRT3, a NAD-dependent deacetylase that affects SDH enzymatic activity [47]; moreover, succinate accumulation in the mitochondrial may increase levels of itaconate, which further inhibit SDH activity [46].…”

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

“…MEPs were prepared from PBMCs using the cold aggregation method with slight modification as described. 28 Briefly, PBMCs were subjected to gentle rocking at 4 C for an hour and aggregated cells that sedimented through fetal bovine serum (FBS; VWR Ò , Radnor, PA) were collected after 8-16 hours for mild monocyte enrichment (»20%¡40% monocytes).…”