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用于测序，并揭示了核糖体的活性翻译。 体内单核苷酸分辨率。它代表了一种有价值的方法来研究蛋白质的各个方面 如翻译效率的调节、选择性翻译起始、核糖体延伸等 以及暂停、密码子使用和鉴定非规范开放阅读框和微肽（<100个氨基酸 酸）在基因组中编码。因此，它提供了翻译控制的独特分子见解，这是以前无法实现的。 可以通过其他基因组技术，如质谱或多核糖体分析来实现。但目前的 核糖体分析方案通常使用复杂的实验程序来分离核糖体-RNA 复合物，如通过蔗糖垫，并需要数百万的输入细胞。这一技术壁垒， 阻止了其应用于检查具有低输入的原始生理组织样本的平移谱 细胞数量。为了解决这个长期存在的挑战，我们建议开发一种超低输入的RNase。 足迹法用于同时快速定量细胞溶质和线粒体翻译。我们 方法简化了基于优化的RNase消化的核糖体足迹选择的实验步骤。 我的实验室已经使这种检测方法在1,000个培养细胞中效果良好。在这项建议中，我们的目标是进一步 开发该检测方法，并使其在少量原代组织样本中稳定工作（目标1）。此外，委员会认为， 我们将应用该方法绘制罕见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）。结果将揭示新的机制介导的翻译控制 造血细胞命运的决定。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，我们将用它来检查线粒体翻译的异质性 机制，并建立由个人因素介导的功能网络（目标3）。总之，精致的超- 本文提出的低输入量的RNA酶足迹法是一种很有价值的方法，具有广泛的应用前景 研究从正常发育到衰老的复杂生理条件下的翻译调节， 疾病进展。

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.