Epitranscriptomic reader YTHDF2 regulates SEK1(MAP2K4)-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress.

As the most abundant glial cells in the central nervous system (CNS), astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress are many and complex. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stressor, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, the neurotoxic stress-induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechanistically, YTHDF2 RIP-sequencing identified MAP2K4 (MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed that Mn-exposed astrocytes mediate proinflammatory responses by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves as a key upstream 'molecular switch' controlling SEK1(MAP2K4)-JNK-cJUN proinflammatory signaling in astrocytes.

m6A RNA methylation regulates mitochondrial function.

RNA methylation of N6-methyladenosine (m6A) is emerging as a fundamental regulator of every aspect of RNA biology. RNA methylation directly impacts protein production to achieve quick modulation of dynamic biological processes. However, whether RNA methylation regulates mitochondrial function is not known, especially in neuronal cells which require a high energy supply and quick reactive responses. Here we show that m6A RNA methylation regulates mitochondrial function through promoting nuclear-encoded mitochondrial complex subunit RNA translation. Conditional genetic knockout of m6A RNA methyltransferase Mettl14 (Methyltransferase like 14) by Nestin-Cre together with metabolomic analysis reveals that Mettl14 knockout-induced m6A depletion significantly downregulates metabolites related to energy metabolism. Furthermore, transcriptome-wide RNA methylation profiling of wild type and Mettl14 knockout mouse brains by m6A-Seq shows enrichment of methylation on mitochondria-related RNA. Importantly, loss of m6A leads to a significant reduction in mitochondrial respiratory capacity and membrane potential. These functional defects are paralleled by the reduced expression of mitochondrial electron transport chain complexes, as well as decreased mitochondrial super-complex assembly and activity. Mechanistically, m6A depletion decreases the translational efficiency of methylated RNA encoding mitochondrial complex subunits through reducing their association with polysomes, while not affecting RNA stability. Together, these findings reveal a novel role for RNA methylation in regulating mitochondrial function. Given that mitochondrial dysfunction and RNA methylation have been increasingly implicate in neurodegenerative disorders, our findings not only provide insights into fundamental mechanisms regulating mitochondrial function, but also open up new avenues for understanding the pathogenesis of neurological diseases.

Protocol for detecting RBM33-binding sites in HEK293T cells using PAR-CLIP-seq.

RNA-binding proteins (RBPs) regulate gene expression both co-transcriptionally and post-transcriptionally. Here, we provide a protocol for photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by next-generation sequencing (PAR-CLIP-seq). PAR-CLIP-seq is a transcriptome-scale technique for identifying in vivo binding sites of RBPs at the single-nucleotide level. We detail procedures for the establishment of FLAG-RBM33 stable cell line, the sequencing library preparation, and the data analysis.

Epitranscriptomic Reader YTHDF2 Regulates SEK1( MAP2K4 )-JNK-cJUN Inflammatory Signaling in Astrocytes during Neurotoxic Stress.

As the most abundant glial cells in the CNS, astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress have remained elusive. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stresser, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, neurotoxic stress induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechnistically, YTHDF2 RIP-sequencing identified MAP2K4 ( MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed Mn-exposed astrocytes mediates proinflammatory response by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves a key upstream 'molecular switch' controlling SEK1( MAP2K4 )-JNK-cJUN proinflammatory signaling in astrocytes.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal-assisted labeling.

Transcription and its dynamics are crucial for gene expression regulation. However, very few methods can directly read out transcriptional activity with low-input material and high temporal resolution. This protocol describes KAS-seq, a robust and sensitive approach for capturing genome-wide single-stranded DNA (ssDNA) profiles using N3-kethoxal-assisted labeling. We developed N3-kethoxal, an azido derivative of kethoxal that reacts with deoxyguanosine bases of ssDNA in live cells within 5-10 min at 37 °C, allowing the capture of dynamic changes. Downstream biotinylation of labeled DNA occurs via copper-free click chemistry. Altogether, the KAS-seq procedure involves N3-kethoxal labeling, DNA isolation, biotinylation, fragmentation, affinity pull-down, library preparation, sequencing and bioinformatics analysis. The pre-library construction labeling and enrichment can be completed in as little as 3-4 h and is applicable to both animal tissue and as few as 1,000 cultured cells. Our recent study shows that ssDNA signals measured by KAS-seq simultaneously reveal the dynamics of transcriptionally engaged RNA polymerase (Pol) II, transcribing enhancers, RNA Pol I and Pol III activities and potentially non-canonical DNA structures with high analytical sensitivity. In addition to the experimental protocol, we also introduce here KAS-pipe, a user-friendly integrative data analysis pipeline for KAS-seq.

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m6A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m6A modification, the methyltransferase responsible for the 18S rRNA m6A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m6A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m6A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m6A in noncoding RNAs.

METTL3 Regulates Liver Homeostasis, Hepatocyte Ploidy, and Circadian Rhythm-Controlled Gene Expression in Mice.

N6-methyladenosine (m6A), the most abundant internal modifier of mRNAs installed by the methyltransferase 13 (METTL3) at the (G/A)(m6A)C motif, plays a critical role in the regulation of gene expression. METTL3 is essential for embryonic development, and its dysregulation is linked to various diseases. However, the role of METTL3 in liver biology is largely unknown. In this study, METTL3 function was unraveled in mice depleted of Mettl3 in neonatal livers (Mettl3fl/fl; Alb-Cre). Liver-specific Mettl3 knockout (M3LKO) mice exhibited global decrease in m6A on polyadenylated RNAs and pathologic features associated with nonalcoholic fatty liver disease (eg, hepatocyte ballooning, ductular reaction, microsteatosis, pleomorphic nuclei, DNA damage, foci of altered hepatocytes, focal lobular and portal inflammation, and elevated serum alanine transaminase/alkaline phosphatase levels). Mettl3-depleted hepatocytes were highly proliferative, with decreased numbers of binucleate hepatocytes and increased nuclear polyploidy. M3LKO livers were characterized by reduced m6A and expression of several key metabolic transcripts regulated by circadian rhythm and decreased nuclear protein levels of the core clock transcription factors BMAL1 and CLOCK. A significant decrease in total Bmal1 and Clock mRNAs but an increase in their nuclear levels were observed in M3LKO livers, suggesting impaired nuclear export. Consistent with the phenotype, methylated (m6A) RNA immunoprecipitation coupled with sequencing and RNA sequencing revealed transcriptome-wide loss of m6A markers and alterations in abundance of mRNAs involved in metabolism in M3LKO. Collectively, METTL3 and m6A modifications are critical regulators of liver homeostasis and function.

Lysine acetylation restricts mutant IDH2 activity to optimize transformation in AML cells.

Mutant isocitrate dehydrogenase (IDH) 1 and 2 play a pathogenic role in cancers, including acute myeloid leukemia (AML), by producing oncometabolite 2-hydroxyglutarate (2-HG). We recently reported that tyrosine phosphorylation activates IDH1 R132H mutant in AML cells. Here, we show that mutant IDH2 (mIDH2) R140Q commonly has K413 acetylation, which negatively regulates mIDH2 activity in human AML cells by attenuating dimerization and blocking binding of substrate (α-ketoglutarate) and cofactor (NADPH). Mechanistically, K413 acetylation of mitochondrial mIDH2 is achieved through a series of hierarchical phosphorylation events mediated by tyrosine kinase FLT3, which phosphorylates mIDH2 to recruit upstream mitochondrial acetyltransferase ACAT1 and simultaneously activates ACAT1 and inhibits upstream mitochondrial deacetylase SIRT3 through tyrosine phosphorylation. Moreover, we found that the intrinsic enzyme activity of mIDH2 is much higher than mIDH1, thus the inhibitory K413 acetylation optimizes leukemogenic ability of mIDH2 in AML cells by both producing sufficient 2-HG for transformation and avoiding cytotoxic accumulation of intracellular 2-HG.

Paraventricular Calcitonin Receptor-Expressing Neurons Modulate Energy Homeostasis in Male Mice.

The paraventricular nucleus of the hypothalamus (PVH) is a heterogeneous collection of neurons that play important roles in modulating feeding and energy expenditure. Abnormal development or ablation of the PVH results in hyperphagic obesity and defects in energy expenditure whereas selective activation of defined PVH neuronal populations can suppress feeding and may promote energy expenditure. Here, we characterize the contribution of calcitonin receptor-expressing PVH neurons (CalcRPVH) to energy balance control. We used Cre-dependent viral tools delivered stereotaxically to the PVH of CalcR2Acre mice to activate, silence, and trace CalcRPVH neurons and determine their contribution to body weight regulation. Immunohistochemistry of fluorescently-labeled CalcRPVH neurons demonstrates that CalcRPVH neurons are largely distinct from several PVH neuronal populations involved in energy homeostasis; these neurons project to regions of the hindbrain that are implicated in energy balance control, including the nucleus of the solitary tract and the parabrachial nucleus. Acute activation of CalcRPVH neurons suppresses feeding without appreciably augmenting energy expenditure, whereas their silencing leads to obesity that may be due in part due to loss of PVH melanocortin-4 receptor signaling. These data show that CalcRPVH neurons are an essential component of energy balance neurocircuitry and their function is important for body weight maintenance. A thorough understanding of the mechanisms by which CalcRPVH neurons modulate energy balance might identify novel therapeutic targets for the treatment and prevention of obesity.

Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m6A Methylation.

N6-methyladenosine (m6A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m6A methylation in mRNA metabolism has been well documented recently, regulation of the m6A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m6A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m6A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m6A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m6A methylation.

Genetic analyses support the contribution of mRNA N6-methyladenosine (m6A) modification to human disease heritability.

N6-methyladenosine (m6A) plays important roles in regulating messenger RNA processing. Despite rapid progress in this field, little is known about the genetic determinants of m6A modification and their role in common diseases. In this study, we mapped the quantitative trait loci (QTLs) of m6A peaks in 60 Yoruba (YRI) lymphoblastoid cell lines. We found that m6A QTLs are largely independent of expression and splicing QTLs and are enriched with binding sites of RNA-binding proteins, RNA structure-changing variants and transcriptional features. Joint analysis of the QTLs of m6A and related molecular traits suggests that the downstream effects of m6A are heterogeneous and context dependent. We identified proteins that mediate m6A effects on translation. Through integration with data from genome-wide association studies, we show that m6A QTLs contribute to the heritability of various immune and blood-related traits at levels comparable to splicing QTLs and roughly half of expression QTLs. By leveraging m6A QTLs in a transcriptome-wide association study framework, we identified putative risk genes of these traits.

RNA Demethylase ALKBH5 Selectively Promotes Tumorigenesis and Cancer Stem Cell Self-Renewal in Acute Myeloid Leukemia.

N6-methyladenosine (m6A), the most abundant internal modification in mRNA, has been implicated in tumorigenesis. As an m6A demethylase, ALKBH5 has been shown to promote the development of breast cancer and brain tumors. However, in acute myeloid leukemia (AML), ALKBH5 was reported to be frequently deleted, implying a tumor-suppressor role. Here, we show that ALKBH5 deletion is rare in human AML; instead, ALKBH5 is aberrantly overexpressed in AML. Moreover, its increased expression correlates with poor prognosis in AML patients. We demonstrate that ALKBH5 is required for the development and maintenance of AML and self-renewal of leukemia stem/initiating cells (LSCs/LICs) but not essential for normal hematopoiesis. Mechanistically, ALKBH5 exerts tumor-promoting effects in AML by post-transcriptional regulation of its critical targets such as TACC3, a prognosis-associated oncogene in various cancers. Collectively, our findings reveal crucial functions of ALKBH5 in leukemogenesis and LSC/LIC self-renewal/maintenance and highlight the therapeutic potential of targeting the ALKBH5/m6A axis.

REPIC: a database for exploring the N6-methyladenosine methylome.

The REPIC (RNA EPItranscriptome Collection) database records about 10 million peaks called from publicly available m6A-seq and MeRIP-seq data using our unified pipeline. These data were collected from 672 samples of 49 studies, covering 61 cell lines or tissues in 11 organisms. REPIC allows users to query N6-methyladenosine (m6A) modification sites by specific cell lines or tissue types. In addition, it integrates m6A/MeRIP-seq data with 1418 histone ChIP-seq and 118 DNase-seq data tracks from the ENCODE project in a modern genome browser to present a comprehensive atlas of m6A methylation sites, histone modification sites, and chromatin accessibility regions. REPIC is accessible at https://repicmod.uchicago.edu/repic.

Lactational metformin exposure programs offspring white adipose tissue glucose homeostasis and resilience to metabolic stress in a sex-dependent manner.

We previously demonstrated that exposing mouse dams to metformin during gestation results in increased beta-cell mass at birth and increased beta-cell insulin secretion in adult male offspring. Given these favorable changes after a gestational maternal metformin exposure, we wanted to understand the long-term metabolic impact on offspring after exposing dams to metformin during the postnatal window. The newborn period provides a feasible clinical window for intervention and is important for beta-cell proliferation and metabolic tissue development. Using a C57BL/6 model, we administered metformin to dams from the day of birth to postnatal day 21. We monitored maternal health and offspring growth during the lactation window, as well as adult glucose homeostasis through in vivo testing. At necropsy we assessed pancreas and adipocyte morphology using histological and immunofluorescent staining techniques. We found that metformin exposure programmed male and female offspring to be leaner with a higher proportion of small adipocytes in the gonadal white adipose tissue (GWAT). Male, but not female, offspring had an improvement in glucose tolerance as young adults concordant with a mild increase in insulin secretion in response to glucose in vivo. These data demonstrate long-term metabolic programming of offspring associated with maternal exposure to metformin during lactation.

N6-Deoxyadenosine Methylation in Mammalian Mitochondrial DNA.

N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.

RADAR: differential analysis of MeRIP-seq data with a random effect model.

Epitranscriptome profiling using MeRIP-seq is a powerful technique for in vivo functional studies of reversible RNA modifications. We develop RADAR, a comprehensive analytical tool for detecting differentially methylated loci in MeRIP-seq data. RADAR enables accurate identification of altered methylation sites by accommodating variability of pre-immunoprecipitation expression level and post-immunoprecipitation count using different strategies. In addition, it is compatible with complex study design when covariates need to be incorporated in the analysis. Through simulation and real dataset analyses, we show that RADAR leads to more accurate and reproducible differential methylation analysis results than alternatives, which is available at https://github.com/scottzijiezhang/RADAR.

The RNA-binding protein FMRP facilitates the nuclear export of N6-methyladenosine-containing mRNAs.

N6-Methyladenosine (m6A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to m6A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m6A sites on mRNAs. FMRP depletion increased mRNA m6A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m6A-modified RNA targets.

Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues.

Spatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reaction (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrate 5- to 180-fold signal amplification in diverse samples (cultured cells, cryosections, formalin-fixed paraffin-embedded sections and whole-mount tissues), as well as simultaneous signal amplification for ten different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.