A large number of nucleus-encoded messenger RNAs (mRNAs) encoding proteins involved in oxidative phosphorylation have been found to be associated with mitochondria in vivo, indicating organelle-specific mRNA targeting. However, the identification of mitochondrion-bound mRNA (Mtb-RNA) has traditionally relied on cumbersome isolations of polysomes from a large number of input cells and is therefore biased in favour of mRNAs associated through nascent targeting peptides emerging from the polysome during cotranslational import of their encoded proteins, and tends to ignore sequence-directed mRNA targeting. We have, therefore, sought to identify and quantify Mtb-RNAs rapidly in small numbers of cells, independently of their polysomal status. We isolated Mtb-RNAs from tissue-cultured cells under different conditions and assayed them by endpoint or real-time polymerase chain reaction (RT-PCR). We observed that (i) different Mtb-RNAs are differentially affected by cycloheximide-induced polysome arrest, indicating possible artifacts of the use of this translation elongation inhibitor; (ii) several Mtb-RNAs have direct affinity for the mitochondrial surface in vitro, indicating the possibility of targeting through mRNA recognition by surface-bound RNA-binding proteins (RBP); and (iii) mRNA–mitochondrion interactions are stabilized by formaldehyde crosslinking. Our results reveal the importance of sequence-directed targeting of mRNAs to mitochondria.

已发现大量核编码的信使 RNA (mRNA) 编码参与氧化磷酸化的蛋白质与体内线粒体有关，表明细胞器特异性 mRNA 靶向。然而，线粒体结合 mRNA (Mtb-RNA) 的鉴定传统上依赖于从大量输入细胞中繁琐地分离多核糖体，因此偏向于通过在共翻译导入过程中从多核糖体中出现的新生靶向肽相关的 mRNA他们编码的蛋白质，并倾向于忽略序列定向的 mRNA 靶向。因此，我们试图在少量细胞中快速识别和量化 Mtb-RNA，而与其多聚体状态无关。我们在不同条件下从组织培养细胞中分离出 Mtb-RNA，并通过终点或实时聚合酶链反应 (RT-PCR) 对其进行测定。我们观察到 (i) 不同的 Mtb-RNA 受到放线菌酮诱导的多核糖体停滞的不同影响，指出使用这种翻译延伸抑制剂可能产生的假象；(ii) 几种 Mtb-RNA 对线粒体表面具有直接亲和力在体外，表明通过表面结合的 RNA 结合蛋白 (RBP) 识别 mRNA 进行靶向的可能性；(iii) mRNA-线粒体相互作用通过甲醛交联得到稳定。我们的结果揭示了 mRNA 序列定向靶向线粒体的重要性。