Shi Yi, Wei Na, Yang Xiang-Lei
Departments of Chemical Physiology and Cell & Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA; The School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China.
Departments of Chemical Physiology and Cell & Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Methods. 2017 Jan 15;113:105-110. doi: 10.1016/j.ymeth.2016.09.011. Epub 2016 Sep 21.
Aminoacyl tRNA synthetases (AARSs) are best known for their essential role in translation in the cytoplasm. The concept that AARSs also exist in the nucleus started to draw attention around the turn of the new millennium, when aminoacylated tRNAs were first found in the nuclei of Xenopus oocytes. It is now expected that all cytoplasmic AARSs are present in the nucleus. In addition to tRNA aminoacylation, nuclear AARSs were found to regulate a spectrum of biological processes and responses, with many AARSs functioning through regulation at the level of gene transcription. In this paper, we focus on describing methods that have been successfully implemented to study AARSs in transcriptional regulation. These include a cell fractionation assay to detect nuclear localization, an in vitro DNA-cellulose pull-down assay to determine DNA binding capacity, and a chromatin immunoprecipitation (ChIP)-DNA deep sequencing assay to identify DNA binding sites. Application of these methods would expand our understanding of AARS functions and reveal critical insights on the coordination of gene transcription and translation.
氨酰-tRNA合成酶（AARSs）因其在细胞质翻译中的关键作用而广为人知。新千年之交，当人们首次在非洲爪蟾卵母细胞核中发现氨酰化tRNA时，AARSs也存在于细胞核中的这一概念开始受到关注。现在预计所有细胞质AARSs都存在于细胞核中。除了tRNA氨酰化作用外，人们发现核AARSs可调节一系列生物过程和反应，许多AARSs通过在基因转录水平上进行调控发挥作用。在本文中，我们重点描述已成功用于研究AARSs转录调控的方法。这些方法包括用于检测核定位的细胞分级分离测定法、用于确定DNA结合能力的体外DNA-纤维素下拉测定法，以及用于识别DNA结合位点的染色质免疫沉淀（ChIP）-DNA深度测序测定法。应用这些方法将扩展我们对AARS功能的理解，并揭示有关基因转录和翻译协调的关键见解。
