N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.
Muscle stem cells are required for the homeostasis and regeneration of mammalian skeletal muscles. It has been reported that RNA N6‐methyladenosine (m6A) modifications play a pivotal role in muscle development and regeneration. Nevertheless, we know little about which m6A reader regulates mammalian muscle stem cells. Here, we discovered that the m6A reader Ythdc1 is indispensable for mouse skeletal muscle regeneration and proliferation of muscle stem cells. In the absence of Ythdc1, Muscle stem cells in adult mice are unable to exit from quiescence. Mechanistically, Ythdc1 binds to m6A‐modified Pi4k2a and Pi4kb mRNAs to regulate their alternative splicing and thus PI4K–Akt–mTOR signalling. Ythdc1‐null muscle stem cells show a deficiency in phosphatidylinositol (PI) 3,4,5‐trisphosphate, phospho‐Akt and phospho‐S6, which correlates with a failure in exit from quiescence. Our findings connect dynamic RNA methylation to the regulation of PI4K–Akt–mTOR signalling during stem cell proliferation and adult tissue regeneration.
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