LRPPRC-mediated folding of the mitochondrial transcriptome [RNase footprinting]. Mus musculus

The roles of RNA-binding proteins as chaperones in the lifecycles of mRNAs are not well understood. The mammalian mitochondrial genome has been compressed over evolution to a size of just 16 kb, nevertheless the expression of its genes requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, providing an opportunity to use it as a model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA-protein interactions guided by the LRPPRC/SLIRP complex in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC/SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization and polyadenylation. Our findings reveal a general mechanism where extensive RNA-protein interactions ensure that RNA is accessible for its biological functions. Overall design: RNase footprinting of LRPPRC and SLIRP knockout and control mice, in technical duplicate.

RNA结合蛋白（RNA-binding protein）作为分子伴侣在信使RNA（mRNA）生命周期中的作用尚未被充分阐明。哺乳动物线粒体基因组经进化压缩至仅16 kb的大小，尽管如此，其基因表达仍需经历转录、RNA加工、翻译及RNA降解等过程，与更为复杂的染色体系统并无二致，这为将其作为模型系统以探究基因表达的核心机制提供了绝佳契机。
本研究将核糖核酸酶足迹法（RNase footprinting）与光活性增强核糖核苷交联免疫沉淀（Photoactivatable-Ribonucleoside Crosslinking and Immunoprecipitation，PAR-CLIP）以空前的实验深度进行整合，揭示了由LRPPRC/SLIRP复合物（LRPPRC/SLIRP complex）介导的RNA-蛋白质相互作用在线粒体转录组内调控RNA折叠过程中的关键作用。
研究表明，LRPPRC与其蛋白伴侣SLIRP形成的复合物可广泛结合于线粒体转录组，对mRNA具有结合偏好性；该复合物的缺失会影响转录组的全部二级结构与稳定性。本研究证实，LRPPRC/SLIRP复合物是一类全局性RNA分子伴侣，能够稳定RNA结构，暴露翻译、维持RNA稳定及多聚腺苷酸化所需的功能位点。
本研究结果揭示了一种通用机制：广泛的RNA-蛋白质相互作用可确保RNA具备满足其生物学功能所需的可及性。
整体实验设计：对LRPPRC与SLIRP敲除小鼠及对照小鼠开展核糖核酸酶足迹分析，设置技术重复两组。