Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes.
Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes.
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作者：
Baysal BE;De Jong K;Liu B;Wang J;Patnaik SK;Wallace PK;Taggart RT
背景。 RNA编辑是一种转录后调节机制，可以改变某些基因的编码序列以响应生理需求。我们之前发现了 C-to-U RNA 编辑（C136U、R46X），它可以使正常稳态外周血单核细胞 (PBMC) 中的一小部分琥珀酸脱氢酶 (SDH；线粒体复合物 II) 亚基 B 基因 (SDHB) mRNA 失活。 SDH 是一种异四聚体肿瘤抑制复合物，当其突变时会导致副神经节瘤，其特征是缺氧诱导途径的组成型激活。在这里，我们研究了 SDHB RNA 编辑的调控、程度和细胞类型起源。方法。我们使用从随机健康血小板捐献者获得的短期培养的PBMC，通过冷聚集进行单核细胞富集，采用新型等位基因特异性定量PCR方法、流式细胞术、免疫细胞分离、基因表达微阵列、数据库分析和高通量RNA测序。结果。虽然未培养的富含单核细胞的 PBMC 中的编辑率较低（平均率 2.0%，范围 0.4%–6.3%，n = 42），但在暴露于 1% 氧张力时（平均率 18.2%，范围 2.8%–49.4%，n = 14）以及在血清存在的常氧巨噬细胞分化过程中（平均率10.1%，范围 2.7%–18.8%，n = 17)。 SDHB RNA 编辑的常氧诱导与培养物中单核细胞密集粘附聚集体的形成有关。 CD14 阳性单核细胞分离使 C136U 转录物的百分比在常氧培养物 (n = 5) 中增加 1.25 倍，在缺氧培养物 (n = 4) 中增加 1.68 倍。 CD14 阴性淋巴细胞没有显示 SDHB 编辑的证据。在增加 RNA 编辑过程中，SDHB 基因组 DNA 保持野生型。微阵列分析显示，随着常氧培养物中编辑率的增加，伤口愈合和免疫反应途径基因的表达发生变化。 SDHB 和 SDHD 转录本的高通量测序证实了常氧培养物中 C136U RNA 编辑的诱导，但没有显示出其他可验证的编码编辑。对来自 Illumina Body Map 的 16 个正常人体组织和来自 ENCODE 项目的代表 23 种不同细胞类型的 45 个样本的 SDHB RNA 序列数据进行分析，证实全血 (1.7%) 和两个原代 CD14+ 单核细胞样本 (1.9% 和 2.6%) 中发生了位点特异性 C136U 编辑。相比之下，其他细胞类型在两个数据库中的 C136U 编辑率平均分别为 0.2% 和 0.1%。结论。这些发现表明，某些基因的C-to-U编码RNA编辑是由生理相关环境因素动态诱导的，并表明通过位点特异性RNA编辑对SDHB的表观遗传下调在单核细胞的缺氧适应中发挥作用。
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.
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影响因子：
3.7
作者：
Baysal, Bora E.
通讯作者：
Baysal, Bora E.
SDH 突变数据库：涉及嗜铬细胞瘤、副神经节瘤和线粒体复合物 II 缺陷的琥珀酸脱氢酶序列变异的在线资源。
影响因子：
--
作者：
Bayley, JP;Devilee, P;Taschner, PEM
通讯作者：
Taschner, PEM
DOI：
10.1126/science.1212795
发表时间：
2012-02-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Garrett S;Rosenthal JJ
通讯作者：
Rosenthal JJ
影响因子：
12.3
作者：
Gentleman RC;Carey VJ;Bates DM;Bolstad B;Dettling M;Dudoit S;Ellis B;Gautier L;Ge Y;Gentry J;Hornik K;Hothorn T;Huber W;Iacus S;Irizarry R;Leisch F;Li C;Maechler M;Rossini AJ;Sawitzki G;Smith C;Smyth G;Tierney L;Yang JY;Zhang J
通讯作者：
Zhang J
影响因子：
4.5
作者：
Kleinman, Claudia L.;Adoue, Veronique;Majewski, Jacek
通讯作者：
Majewski, Jacek