PDF(Pubmed)
Abstract：
Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed in cellulo RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2\'s cofactor) were pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2-binding RNA in the cytosol.
摘要：
已经确定了几种中间代谢酶结合2细胞中的RNA，对结合的RNA和/或酶有潜在的影响。在这项研究中，我们研究了线粒体酶苹果酸4脱氢酶2（MDH2）的RNA结合活性，在三羧酸（TCA）循环和苹果酸-天冬氨酸穿梭中起作用。我们使用6种正交生化测定法证实了MDH2的celluloRNA结合，并进行了增强的交联和7种免疫沉淀（eCLIP），以鉴定与内源性8MDH2相关的细胞RNA。令人惊讶的是,MDH2优先结合胞质而不是线粒体RNA，9，尽管后者在成熟蛋白质的环境中丰富。亚细胞分级分离后的RNA结合测定显示,MDH2-RNA相互作用11主要发生在线粒体之外。我们还发现，MDH2的胞质12保留的N端缺失突变体能够结合RNA，表明13线粒体靶向对于MDH2-RNA相互作用是不必要的。当细胞NAD+水平（MDH2的辅因子）在药理学上减少时，MDH2RNA14结合增加，这表明细胞的代谢状态会影响RNA16的结合。一起来看,我们的数据暗示细胞溶质中MDH217结合RNA的功能尚未确定.
