Grelet Simon, McShane Ariel, Hok Eveline, Tomberlin Jensen, Howe Philip H, Geslain Renaud
Department of Biochemistry and Molecular Biology, MUSC, Charleston, SC, United States of America.
Laboratory of tRNA Biology, Department of Biology, College of Charleston, Charleston, SC, United States of America.
PLoS One. 2017 May 17;12(5):e0177939. doi: 10.1371/journal.pone.0177939. eCollection 2017.
Recent studies have placed transfer RNA (tRNA), a housekeeping molecule, in the heart of fundamental cellular processes such as embryonic development and tumor progression. Such discoveries were contingent on the concomitant development of methods able to deliver high-quality tRNA profiles. The present study describes the proof of concept obtained in Escherichia coli (E. coli) for an original tRNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). This approach comprises three steps. First, E. coli cultures are spiked with radioactive orthophosphate; second, labeled total RNAs are trizol-extracted; third, RNA samples are hybridized on in-house printed microarrays and spot signals, the proxy for tRNA levels, are quantified by phosphorimaging. Features such as reproducibility and specificity were assessed using several tRNA subpopulations. Dynamic range and sensitivity were evaluated by overexpressing specific tRNA species. SPOt does not require any amplification or post-extraction labeling and can be adapted to any organism. It is modular and easily streamlined with popular techniques such as polysome fractionation to profile tRNAs interacting with ribosomes and actively engaged in translation. The biological relevance of these data is discussed in regards to codon usage, tRNA gene copy number, and position on the genome.
最近的研究已将转运RNA（tRNA）这种管家分子置于胚胎发育和肿瘤进展等基本细胞过程的核心位置。这些发现取决于能够提供高质量tRNA谱的方法的同步发展。本研究描述了在大肠杆菌（E. coli）中针对一个名为SPOt（观察tRNA的简化平台）的原始tRNA分析平台所获得的概念验证。该方法包括三个步骤。首先，在大肠杆菌培养物中加入放射性正磷酸盐；其次，用三氯甲烷提取标记的总RNA；第三，将RNA样品与自制的微阵列杂交，并通过磷光成像对作为tRNA水平指标的斑点信号进行定量。使用几个tRNA亚群评估了诸如重现性和特异性等特征。通过过表达特定的tRNA种类评估了动态范围和灵敏度。SPOt不需要任何扩增或提取后标记，并且可以适用于任何生物体。它具有模块化特点，并且可以很容易地与诸如多核糖体分级分离等常用技术相结合，以分析与核糖体相互作用并积极参与翻译的tRNA。针对密码子使用、tRNA基因拷贝数以及在基因组上的位置，讨论了这些数据的生物学相关性。
