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

摘要：“表位转录组”，即受调控的核苷酸修饰的模式和分布。 信使核糖核酸，是动态的，可以被环境刺激和诱导细胞应激的物质调节。然而， 量化细胞rna样本中m6A变化的能力是具有挑战性的，因为在 细胞来源于核糖体RNA(RRNA)和U6小核RNA(SnRNA)。因此，mRNA源的M6A 水平可能会在不同的治疗方法之间发生变化，但在某些情况下，这些变化已经 归因于样品中不同程度的rRNA/SnRNA污染。因此，需要一种方法来 使研究人员能够明确地量化细胞mRNA中的m6A水平，并确定它们是否 根据任何细胞环境或实验条件而变化的。在这份提案中，我们描述了“Pho-- M6A分析“--一种区分mRNA源m6A和m6A的磷酸选择性m6A标记方法 RRNA/SnRNA衍生的M6A。Pho-m6A检测使用了一套从mRNA中产生m6A的酶步骤 和来自污染物的m6A用不同的磷酸盐标记-来自mRNA的m6A没有磷酸盐，以及 一种M6A的单一磷酸盐，可防止污染RNA。Pho-M6A提供了高度简化但高度 可用于细胞总RNA的信使核糖核酸M6A的精确测定，而无论是否存在 污染rRNA和SnRNA。Pho-M6A分析使我们能够解决与以下哪些关键问题相关的问题 细胞应激和应激诱导剂调节细胞内的m6A水平。为了显著推进我们的 能够量化和测量由细胞外应激源诱导的m6A的动力学，其具体目标是 建议：(1)优化Pho-m6A法检测mRNA中m6A的方法。在这个目标中，我们将优化 Pho-m6A检测侧重于确定我们的检测的绝对灵敏度(即RNA输入水平)， 可以准确量化的m6A的绝对差异，并确定噪声和变异性 确定准确测量信使核糖核酸样本中m6A的重复次数。 这些实验将建立使用Pho-m6A测定m6A的最佳条件 在任何RNA样本中的mRNA的动力学。(2)建立暴露后m6A在mRNA中的动态变化。 环境刺激。为此，我们将使用Pho-m6A分析来发现m6A变化的时间进程 在细胞应激后的mRNA中，以及筛选各种环境刺激 与转录组中的m6A改变有关。根据时间进程，我们将使用时间点 其中m6A最大限度地被激活，用于转录组范围的m6A图谱研究。总体而言，我们预计 PHO-M6A检测将克服因污染而引起的现场可变性和不一致性 RRNA/SnRNA。这些研究也将为理解两者之间的联系提供重要的基础 细胞应激与表位转录组的动态调控 M6A，以及应激诱导的特定M6A位点。

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.