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

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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.