Pho-m6A assay: A phosphoselective method to quantify dynamics of m6A in mRNA

Pho-m6A 测定：一种定量 mRNA 中 m6A 动态的磷酸选择性方法

• 批准号: 10271260
• 负责人: Qiuying Chen
• 金额: $ 25.43万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-28 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10271260
• 关键词: Address; Biological Assay; Cells; Cellular Stress; Complex; Detection; Enzymes; Exposure to; Foundations; GTP-Binding Protein alpha Subunits, Gs; Heat-Shock Response; Isotope Labeling; Libraries; Link; Mass Spectrum Analysis; Measurement; Measures; Mediating; Messenger RNA; Methods; Modification; Noise; Nucleotides; Pathway interactions; Pattern; Preparation; Proxy; RNA; Reaction; Regulation; Reproducibility; Research Personnel; Ribosomal RNA; Role; Sampling; Signal Transduction; Signaling Molecule; Site; Small Nuclear RNA; Stimulus; Stress; Techniques; Time; Toxic Environmental Substances; Toxin; Transcript; U6 small nuclear RNA; Ultraviolet Rays; base; cell injury; epitranscriptome; experimental study; extracellular; inorganic phosphate; mRNA capping; next generation sequencing; response; screening; stressor; time use; transcriptome

SUMMARY: The “epitranscriptome,” i.e., the pattern and distribution of regulated nucleotide modifications in mRNA, is dynamic and can be regulated by environmental stimuli and agents that induce cell stress. However, the ability to quantify m6A changes in cellular RNA samples is challenging since the vast majority of m6A in a cell derives from ribosomal RNA (rRNA) and the U6 small nuclear RNA (snRNA). Thus, mRNA-derived m6A levels may appear to change between different treatments, but in some cases these changes have been attributed to different levels of contaminating rRNA/snRNA in the samples. Thus, a method is needed that would allow researchers to unambiguously quantify m6A levels in cellular mRNA and to determine if they are changing in response to any cellular context or experimental condition. In this proposal, we describe the “Pho- m6A assay” – a phosphate-selective m6A tagging approach to distinguish mRNA-derived m6A from rRNA/snRNA-derived m6A. The Pho-m6A assay uses a set of enzymatic steps that causes m6A from mRNA and m6A from contaminants to be differently marked with phosphates – no phosphate for m6A from mRNA, and a single phosphate for m6A from contaminating RNA. The Pho-m6A provides a highly simplified, yet highly precise assay for mRNA-derived m6A that can be used on total cellular RNA, regardless of the presence of contaminating rRNA and snRNA. The Pho-m6A assay allows us to address critical questions related to which cellular stresses and stress-inducing agents regulate m6A levels in cells. In order to significantly advance our ability to quantify and measure the dynamics of m6A induced by extracellular stressors, the specific aims of this proposal are: (1) To optimize the detection of m6A in mRNA by the Pho-m6A assay. In this aim, we will optimize the Pho-m6A assay focusing on establishing the absolute sensitivity of our assay (i.e. RNA input levels), the absolute differences in m6A that can be accurately quantified, and determining the noise and variability of the assay to determine the number of replicates required for accurate measurements of m6A in mRNA samples. These experiments will establish the optimal conditions for using the Pho-m6A assay to measure m6A dynamics in mRNA in any RNA sample. (2) To establish the dynamics of m6A in mRNA after exposure to environmental stimuli. In this aim, we will use the Pho-m6A assay to discover the time course of m6A changes in mRNA after cell stress, as well as to screen various environmental stimuli to discover which ones are associated with m6A alterations in the transcriptome. Based on the time course, we will use the time points where m6A is maximally activated for transcriptome-wide m6A mapping studies. Overall, we expect that the Pho-m6A assay will overcome the variability and inconsistencies in the field caused by contaminating rRNA/snRNA. These studies will also provide an important foundation for understanding the links between cellular stress and dynamic regulation of the epitranscriptome by identifying the specific stressors that induce m6A, and the specific m6A sites that are induced by stress.

总结：“表转录组”，即，受调控的核苷酸修饰的模式和分布， mRNA是动态的，并且可以通过诱导细胞应激的环境刺激和试剂来调节。然而，在这方面， 定量细胞RNA样品中m6 A变化的能力是具有挑战性的， 细胞中的核糖体RNA（rRNA）和U6小核RNA（snRNA）。因此，mRNA衍生的m6 A 水平可能会出现不同的治疗之间的变化，但在某些情况下，这些变化已经 这归因于样品中不同水平的污染rRNA/snRNA。因此，需要一种方法， 这将使研究人员能够明确地量化细胞mRNA中的m6 A水平，并确定它们是否 对任何细胞环境或实验条件的反应而改变。在这一建议中，我们描述了“Pho- m6 A测定”-一种磷酸选择性m6 A标记方法，用于区分mRNA衍生的m6 A与 rRNA/snRNA衍生的m6 A。Pho-m6 A检测使用一组酶促步骤， 和来自污染物的m6 A用磷酸盐不同地标记-来自mRNA的m6 A没有磷酸盐，和 从污染的RNA中提取m6 A的单磷酸盐。Pho-m6 A提供了一个高度简化，但高度 可用于总细胞RNA的mRNA衍生m6 A的精确测定，无论是否存在 污染rRNA和snRNA。Pho-m6 A检测使我们能够解决与以下方面相关的关键问题： 细胞应激和应激诱导剂调节细胞中的m6 A水平。为了大大提高我们的 定量和测量由细胞外应激源诱导的m6 A动力学的能力，本发明的具体目的是 （1）优化Pho-m6 A法检测mRNA中m6 A的方法。为此，我们将优化 Pho-m6 A测定侧重于确定我们测定的绝对灵敏度（即RNA输入水平）， 可以准确量化的m6 A的绝对差异，并确定m6 A的噪声和变异性。 测定以确定精确测量mRNA样品中m6 A所需的重复次数。 这些实验将建立使用Pho-m6 A测定法测量m6 A的最佳条件 在任何RNA样品中的mRNA动力学。(2)为了建立暴露后m6 A在mRNA中的动力学， 环境刺激为此，我们将使用Pho-m6 A测定来发现m6 A变化的时间过程 以及筛选各种环境刺激，以发现哪些是 与转录组中的m6 A改变相关。根据时间进程，我们将使用时间点 其中m6 A被最大程度地激活用于转录组范围m6 A作图研究。总的来说，我们预计， Pho-m6 A检测试剂盒将克服污染导致的现场变异性和不一致性 rRNA/snRNA。这些研究还将为理解 细胞应激和动态调节的表位转录组通过鉴定特定的应激源， m6 A，以及由应力诱导的特定m6 A位点。