Quantitative profiling of m6A at single base resolution across the life cycle of rice and Arabidopsis.

N6-methyladenosine (m6A) plays critical roles in regulating mRNA metabolism. However, comprehensive m6A methylomes in different plant tissues with single-base precision have yet to be reported. Here, we present transcriptome-wide m6A maps at single-base resolution in different tissues of rice and Arabidopsis using m6A-SAC-seq. Our analysis uncovers a total of 205,691 m6A sites distributed across 22,574 genes in rice, and 188,282 m6A sites across 19,984 genes in Arabidopsis. The evolutionarily conserved m6A sites in rice and Arabidopsis ortholog gene pairs are involved in controlling tissue development, photosynthesis and stress response. We observe an overall mRNA stabilization effect by 3' UTR m6A sites in certain plant tissues. Like in mammals, a positive correlation between the m6A level and the length of internal exons is also observed in plant mRNA, except for the last exon. Our data suggest an active m6A deposition process occurring near the stop codon in plant mRNA. In addition, the MTA-installed plant mRNA m6A sites correlate with both translation promotion and translation suppression, depicting a more complicated regulatory picture. Our results therefore provide in-depth resources for relating single-base resolution m6A sites with functions in plants and uncover a suppression-activation model controlling m6A biogenesis across species.

Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation.

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

Galectin-3 Mediates Endotoxin Internalization and Caspase-4/11 Activation in Tubular Epithelials and Macrophages During Sepsis and Sepsis-Associated Acute Kidney Injury.

Besides being recognized by membrane receptor TLR4, lipopolysaccharide (LPS) can also be internalized into the cytosol and activate Caspase-4/11 pyroptotic pathways to further amplify inflammation in sepsis. The objective of this study was to investigate whether Galectin-3 (Gal3) could promote the uptake of LPS by governing RAGE or administering endocytosis, consequently activating Caspase 4/11 and mediating pyroptosis in sepsis-associated acute kidney injury (SA-AKI). By pinpointing Gal3, LPS, and EEA1 (endosome-marker) or LAMP1 (lysosome-marker) respectively, immunofluorescence discovered that Gal3 and LPS were mainly aggregated in early endosomes initially and translocated into lysosomes afterwards. In cells and animal models, Gal3 and the Caspase-4/11 pathways were simultaneously activated, and the overexpression of Gal3 could exacerbate pyroptosis, whereas inhibition of Gal3 or the knockdown of its expression could ameliorate pyroptosis, reduce the pathological changes of SA-AKI and improve the survival of the animals with SA-AKI. Silencing RAGE reduced pyroptosis in primary tubular epithelial cells (PTCs) activated by Gal3 and LPS but not in cells activated by Gal3 and outer membrane vesicles (with LPS inside), whereas pyroptosis in both was reduced by blockade of Gal3, indicating Gal3 promoted pyroptosis through both RAGE-dependent and RAGE-independent pathways. Our investigation further revealed a positive correlation between serum Gal3 and pyroptotic biomarkers IL-1 beta and IL-18 in patients with sepsis, and that serum Gal3 was an independent risk factor for mortality. Through our collective exploration, we unraveled the significant role of Gal3 in the internalization of LPS and the provocation of more intense pyroptosis, thus making it a vital pathogenic factor in SA-AKI and a possible therapeutic target. Gal3 enabled the internalization of endotoxin into endosomes and lysosomes via both RAGE-dependent (A) and RAGE-independent (B) pathways, leading to pyroptosis. The suppression of Gal3 curbed Caspase4/11 noncanonical inflammasomes and diminished sepsis and SA-AKI.

Advances in Biomarkers for Diagnosis and Treatment of ARDS.

Acute respiratory distress syndrome (ARDS) is a common and fatal disease, characterized by lung inflammation, edema, poor oxygenation, and the need for mechanical ventilation, or even extracorporeal membrane oxygenation if the patient is unresponsive to routine treatment. In this review, we aim to explore advances in biomarkers for the diagnosis and treatment of ARDS. In viewing the distinct characteristics of each biomarker, we classified the biomarkers into the following six categories: inflammatory, alveolar epithelial injury, endothelial injury, coagulation/fibrinolysis, extracellular matrix turnover, and oxidative stress biomarkers. In addition, we discussed the potential role of machine learning in identifying and utilizing these biomarkers and reviewed its clinical application. Despite the tremendous progress in biomarker research, there remain nonnegligible gaps between biomarker discovery and clinical utility. The challenges and future directions in ARDS research concern investigators as well as clinicians, underscoring the essentiality of continued investigation to improve diagnosis and treatment.

RBFOX2 recognizes N6-methyladenosine to suppress transcription and block myeloid leukaemia differentiation.

N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. However, the mechanism by which MTC is recruited to distinct genomic loci remains elusive. Here we identify RBFOX2, a well-studied RNA-binding protein, as a chromatin factor that preferentially recognizes m6A on caRNAs. RBFOX2 can recruit RBM15, an MTC component, to facilitate methylation of promoter-associated RNAs. RBM15 also physically interacts with YTHDC1 and recruits polycomb repressive complex 2 (PRC2) to the RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this RBFOX2/m6A/RBM15/YTHDC1/PRC2 axis plays a critical role in myeloid leukaemia. Downregulation of RBFOX2 notably inhibits survival/proliferation of acute myeloid leukaemia cells and promotes their myeloid differentiation. RBFOX2 is also required for self-renewal of leukaemia stem/initiation cells and acute myeloid leukaemia maintenance. Our study presents a pathway of m6A MTC recruitment and m6A deposition on caRNAs, resulting in locus-selective chromatin regulation, which has potential therapeutic implications in leukaemia.

Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination.

N6-methyladenosine (m6A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m6A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Here we present evolved TadA-assisted N6-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m6A by global adenosine deamination. With eTAM-seq, we analyze the transcriptome-wide distribution of m6A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m6A detection from limited input samples. In addition to transcriptome-wide m6A profiling, we demonstrate site-specific, deep-sequencing-free m6A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. We envision that eTAM-seq will enable researchers to not only survey the m6A landscape at unprecedented resolution, but also detect m6A at user-specified loci with a simple workflow.

Exon architecture controls mRNA m6A suppression and gene expression.

N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m6A (MPm6A), we discover that m6A specificity is globally regulated by suppressors that prevent m6A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m6A suppressors that protect exon junction-proximal RNA within coding sequences from methylation and regulate mRNA stability through m6A suppression. EJC suppression of m6A underlies multiple global characteristics of mRNA m6A specificity, with the local range of EJC protection sufficient to suppress m6A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes.

m6A-SAC-seq for quantitative whole transcriptome m6A profiling.

N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, regulating many physiological processes. Here we describe a method for base-resolution, quantitative m6A sequencing in the whole transcriptome. The enzyme and small-molecule cofactor used in this protocol are prepared by recombinant protein expression and organic synthesis, respectively. Then the library can be prepared from various types of RNA samples using a ligation-based strategy, with m6A modifications being labeled by the enzyme and cofactor. Detailed instructions on ensuing data analysis are also included in this protocol. The method generates highly reproducible results, uncovering 31,233-129,263 sites using as little as 2 ng of poly A+ RNA. These identified sites correspond well with previous m6A profiling results, covering over 65% of peaks detected by the antibody-based approaches. Compared with other currently available methods, this method can be applied to various types of biological samples, including fresh and frozen tissues as well as formalin-fixed paraffin-embedded samples, providing a quantitative method to uncover new insights into m6A biology. The protocol requires basic expertise in molecular biology, recombinant protein expression and organic synthesis. The whole protocol can be done in 15 days, with the library preparation taking 5 days.

Detection of m6A RNA modifications at single-nucleotide resolution using m6A-selective allyl chemical labeling and sequencing.

As the most abundant internal mRNA modification, N6-methyladenosine (m6A) was involved in almost all the aspects of RNA metabolism. Here, we introduce our protocol for m6A-SAC-seq, which enables the whole transcriptome-wide mapping of m6A RNA modification at single-nucleotide resolution with stoichiometry information. m6A-SAC-seq relies on selective allyl labeling of m6A by specific methyltransferase and chemical treatment that introduce mutation upon reverse transcription. The technique only requires ∼30 ng of input RNA. For complete details on the use and execution of this protocol, please refer to Hu et al. (2022).

m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome.

Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.

Cross-Linked Regulation of Coral-Associated Dinoflagellates and Bacteria in Pocillopora sp. during High-Temperature Stress and Recovery.

As the problem of ocean warming worsens, the environmental adaptation potential of symbiotic Symbiodiniaceae and bacteria is directly related to the future and fate of corals. This study aimed to analyse the comprehensive community dynamics and physiology of these two groups of organisms in the coral Pocillopora sp. through indoor simulations of heat stress (which involved manually adjusting the temperature between both 26 °C and 34 °C). Heat treatment (≥30 °C) significantly reduced the abundance of Symbiodiniaceae and bacteria by more than 70%. After the temperature was returned to 26 °C for one month, the Symbiodiniaceae density was still low, while the absolute number of bacteria quickly recovered to 55% of that of the control. At this time point, the Fv/Fm value rose to 91% of the pretemperature value. The content of chlorophyll b associated with Cyanobacteria increased by 50% compared with that under the control conditions. Moreover, analysis of the Symbiodiniaceae subclade composition suggested that the relative abundance of C1c.C45, C1, and C1ca increased during heat treatment, indicating that they might constitute heat-resistant subgroups. We suggest that the increase in the absolute number of bacteria during the recovery period could be an important indicator of coral holobiont recovery after heat stress. This study provides insight into the cross-linked regulation of key symbiotic microbes in the coral Pocillopora sp. during high-temperature stress and recovery and provides a scientific basis for exploring the mechanism underlying coral adaptation to global warming.

Regulation of the Coral-Associated Bacteria and Symbiodiniaceae in Acropora valida Under Ocean Acidification.

Ocean acidification is one of many stressors that coral reef ecosystems are currently contending with. Thus, understanding the response of key symbiotic microbes to ocean acidification is of great significance for understanding the adaptation mechanism and development trend of coral holobionts. Here, high-throughput sequencing technology was employed to investigate the coral-associated bacteria and Symbiodiniaceae of the ecologically important coral Acropora valida exposed to different pH gradients. After 30 days of acclimatization, we set four acidification gradients (pH 8.2, 7.8, 7.4, and 7.2, respectively), and each pH condition was applied for 10 days, with the whole experiment lasting for 70 days. Although the Symbiodiniaceae density decreased significantly, the coral did not appear to be bleached, and the real-time photosynthetic rate did not change significantly, indicating that A. valida has strong tolerance to acidification. Moreover, the Symbiodiniaceae community composition was hardly affected by ocean acidification, with the C1 subclade (Cladocopium goreaui) being dominant among the Symbiodiniaceae dominant types. The relative abundance of the Symbiodiniaceae background types was significantly higher at pH 7.2, indicating that ocean acidification might increase the stability of the community composition by regulating the Symbiodiniaceae rare biosphere. Furthermore, the stable symbiosis between the C1 subclade and coral host may contribute to the stability of the real-time photosynthetic efficiency. Finally, concerning the coral-associated bacteria, the stable symbiosis between Endozoicomonas and coral host is likely to help them adapt to ocean acidification. The significant increase in the relative abundance of Cyanobacteria at pH 7.2 may also compensate for the photosynthesis efficiency of a coral holobiont. In summary, this study suggests that the combined response of key symbiotic microbes helps the whole coral host resist the threats of ocean acidification.

Multi-Omics Revealing the Response Patterns of Symbiotic Microorganisms and Host Metabolism in Scleractinian Coral Pavona minuta to Temperature Stresses.

Global climate change has resulted in large-scale coral reef decline worldwide, for which the ocean warming has paid more attention. Coral is a typical mutually beneficial symbiotic organism with diverse symbiotic microorganisms, which maintain the stability of physiological functions. This study compared the responses of symbiotic microorganisms and host metabolism in a common coral species, Pavona minuta, under indoor simulated thermal and cold temperatures. The results showed that abnormal temperature stresses had unfavorable impact on the phenotypes of corals, resulting in bleaching and color change. The compositions of symbiotic bacteria and dinoflagellate communities only presented tiny changes under temperature stresses. However, some rare symbiotic members have been showed to be significantly influenced by water temperatures. Finally, by using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS) method, we found that different temperature stresses had very different impacts on the metabolism of coral holobiont. The thermal and cold stresses induced the decrease of anti-oxidation metabolites, several monogalactosyldiacylglycerols (MGDGs), and the increase of lipotoxic metabolite, 10-oxo-nonadecanoic acid, in the coral holobiont, respectively. Our study indicated the response patterns of symbiotic microorganisms and host metabolism in coral to the thermal and cold stresses, providing theoretical data for the adaptation and evolution of coral to a different climate in the future.