An ultra-low-input RNase footprinting assay to quantify cytosolic and mitochondrial translation simultaneously

摘要 核糖体分析分离核糖体保护的片段进行测序，并揭示在 体内单核苷酸拆分。这是一种研究蛋白质各个方面的有价值的方法。 合成，如翻译效率的调节，选择性翻译的启动，核糖体的延长 以及暂停、密码子使用和识别非规范的开放阅读框架和微肽(&lt；100个氨基酸 酸)编码在基因组中的。因此，它提供了独特的翻译控制的分子洞察力，这是不可能的 可通过其他基因组技术实现，例如质谱仪或多聚体图谱。但是，当前 核糖体分析方法通常使用复杂的实验程序来分离核糖体-RNA 复合体如蔗糖般缓冲，并需要数百万个输入细胞。这一技术障碍具有 阻止了其在低输入的初级生理组织样本的平移轮廓检查中的应用 手机号码。为了应对这一长期存在的挑战，我们建议开发一种超低投入的核糖核酸酶 同时对胞质和线粒体翻译进行快速定量的足迹方法。我们的 方法简化基于优化核糖酶消化的核糖体足迹选择实验程序。 我的实验室已经使这项测试在1000个培养细胞中发挥了很好的作用。在这项建议中，我们的目标是进一步 发展这种分析方法，使其对少量的初级组织样本有效(目标1)。此外， 我们将应用该方法绘制稀有祖细胞和分化细胞的RNA翻译图谱 造血过程中的类型(目标2)。研究结果将揭示翻译控制的新机制 潜在的造血细胞命运决定。最后，通过利用我们的分析提供了一种简化的方法 为了研究线粒体翻译，我们将用它来检验线粒体翻译的异质性 机械和建立由个人因素调节的功能网络(目标3)。总而言之，精致的超 该方案开发的低投入的RNase足迹方法将是一个有价值的工具，可以广泛应用 研究从正常发育到复杂生理条件下的翻译调节 疾病的发展。

ABSTRACT Ribosome profiling isolates ribosome-protected fragments for sequencing and reveals active translation at the single-nucleotide resolution in vivo. It represents a valuable approach to study various aspects of protein synthesis, such as the regulation of translation efficiency, alternative translation initiation, ribosome elongation and pausing, codon usage, and identifying non-canonical open reading frames and micropeptides (<100 amino acids) encoded in a genome. Thus, it provides unique molecule insights of translational control, which could not be achieved by other genomic technologies, such as mass spectrometry or polysome profiling. However, current ribosome profiling protocols generally use complicated experimental procedures to isolate ribosome-RNA complexes such as though a sucrose cushion, and require millions of input cells. This technical barrier has prevented its application to examing translation profiles of primary physiological tissue samples with low-input cell numbers. To tackle this long-standing challenge, here we propose to develop an ultra-low-input RNase footprinting approach for the rapid quantification of cytosolic and mitochondrial translation simultaneously. Our method simplified the experimental procedure to select ribosome footprints based on optimized RNase digestion. My lab has made the assay work well for as few as 1,000 cultured cells. In this proposal, we aim to further develop the assay and make it work robustly for a small amount of primary tissue samples (Aim 1). Furthermore, we will apply the method to map the RNA translation landscape of rare progenitor cells and differentiated cell types during hematopoiesis (Aim 2). The results will reveal novel mechanisms mediating the translational control underlying hematopoietic cell fate decisions. Finally, by leveraging that our assay provides a simplified method to study mitochondrial translation, we will use it to examine the heterogeneity of mitochondrial translation machinery and build the functional network mediated by individual factors (Aim 3). Altogether, the refined ultra- low-input RNase footprinting method developed in this proposal will be a valuable tool and can be widely used to study the translational regulation underlying complex physiological conditions from normal development to disease progression.