Deciphering m6A dynamics at a single-base level during planarian anterior-posterior axis specification.

Background: The establishment of the anterior-posterior (A-P) axis is a crucial step during tissue repair and regeneration. Despite the association reported recently of N6-methyladenosine (m6A) with regeneration, the mechanism underlying the regulation of m6A in A-P axis specification during regeneration remains unknown. Herein, we deciphered the m6A landscape at a single-base resolution at multiple time points during A-P axis regeneration and constructed the de novo transcriptome assembly of the Dugesia japonica planarian. Results: Immunofluorescence staining and comparative analysis revealed that m6A is widespread across the planarian and dynamically regulated during regeneration along the A-P axis, exhibiting a strong spatiotemporal feature. The resulting datasets of m6A-modified genes identified 80 anterior-specific genes and 13 posterior-specific genes, respectively. In addition, we showed that YTHDC1 serves as the primary m6A reader to be involved in the m6A-mediated specification of A-P axis during regeneration in Dugesia japonica planarian. Conclusions: Our study provides an RNA epigenetic explanation for the specification of the A-P axis during tissue regeneration in planarian.

The chronic stress risk phenotype mirrored in the human retina as a neurodegenerative condition.

The brain is the key organ that orchestrates the stress response which translates to the retina. The retina is an extension of the brain and retinal symptoms in subjects with neurodegenerative diseases substantiated the eye as a window to the brain. The retina is used in this study to determine whether chronic stress reflects neurodegenerative signs indicative of neurodegenerative conditions. A three-year prospective cohort (n = 333; aged 46 ± 9 years) was stratified into stress-phenotype cases (n = 212) and controls (n = 121) by applying the Malan stress-phenotype index. Neurodegenerative risk markers included ischemia (astrocytic S100 calcium-binding protein B/S100B); 24-h blood pressure, proteomics; inflammation (tumor-necrosis-factor-α/TNF-α); neuronal damage (neuron-specific-enolase); anti-apoptosis of retinal-ganglion-cells (beta-nerve-growth-factor), astrocytic activity (glial-fibrillary-acidic-protein); hematocrit (viscosity) and retinal follow-up data [vessels; stress-optic-neuropathy]. Stress-optic-neuropathy risk was calculated from two indices: a newly derived diastolic-ocular-perfusion-pressure cut-point ≥68 mmHg relating to the stress-phenotype; combined with an established cup-to-disk ratio cut-point ≥0.3. Higher stress-optic-neuropathy (39% vs. 17%) and hypertension (73% vs. 16%) prevalence was observed in the stress-phenotype cases vs. controls. Elevated diastolic-ocular-perfusion-pressure, indicating hypoperfusion, was related to arterial narrowing and trend for ischemia increases in the stress-phenotype. Ischemia in the stress-phenotype at baseline, follow-up and three-year changes was related to consistent inflammation (TNF-α and cytokine-interleukin-17-receptor-A), neuron-specific-enolase increases, consistent apoptosis (chitinase-3-like protein 1, low beta-nerve-growth-factor), glial-fibrillary-acidic-protein decreases, elevated viscosity, vein widening as risk marker of endothelial dysfunction in the blood-retinal barrier, lower vein count, and elevated stress-optic-neuropathy. The stress-phenotype and related neurodegenerative signs of ongoing brain ischemia, apoptosis and endothelial dysfunction compromised blood-retinal barrier permeability and optic nerve integrity. In fact, the stress-phenotype could identify persons at high risk of neurodegeneration to indicate a neurodegenerative condition.

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing.

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.

High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome.

N6-deoxyadenosine methylation (6mA) is the most widespread type of DNA modification in prokaryotes and is also abundantly distributed in some unicellular eukaryotes. However, 6mA levels are remarkably low in mammals. The lack of a precise and comprehensive mapping method has hindered more advanced investigations of 6mA. Here, we report a new method MM-seq (modification-induced mismatch sequencing) for genome-wide 6mA mapping based on a novel detection principle. We found that modified DNA bases are prone to form a local open region that allows capture by antibody, for example, via a DNA breathing or base-flipping mechanism. Specified endonuclease or exonuclease can recognize the antibody-stabilized mismatch-like structure and mark the exact modified sites for sequencing readout. Using this method, we examined the genomic positions of 6mA in bacteria (E. coli), green algae (C. reinhardtii), and mammalian cells (HEK239T, Huh7, and HeLa cells). In contrast to bacteria and green algae, human cells possess a very limited number of 6mA sites which are sporadically distributed across the genome of different cell types. After knocking out the RNA m6A methyltransferase METTL3 in mouse ES cells, 6mA becomes mostly diminished. Our results imply that rare 6mA in the mammalian genome is introduced by RNA m6A machinery via a non-targeted mechanism.