R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling.

R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling.

N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in mammalian mRNA. This modification is reversible and non-stoichiometric and adds another layer to the dynamic control of mRNA metabolism. The stability of m(6)A-modified mRNA is regulated by an m(6)A reader protein, human YTHDF2, which recognizes m(6)A and reduces the stability of target transcripts. Looking at additional functional roles for the modification, we find that another m(6)A reader protein, human YTHDF1, actively promotes protein synthesis by interacting with translation machinery. In a unified mechanism of m(6)A-based regulation in the cytoplasm, YTHDF2-mediated degradation controls the lifetime of target transcripts, whereas YTHDF1-mediated translation promotion increases translation efficiency, ensuring effective protein production from dynamic transcripts that are marked by m(6)A. Therefore, the m(6)A modification in mRNA endows gene expression with fast responses and controllable protein production through these mechanisms.

N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.

N(6)-methyldeoxyadenosine (6mA or m(6)A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here, we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms.

A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution.

Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE. pseU-TRACE relied on the fact that RNA containing Ψ underwent a base deletion after treatment of bisulfite (BS) during reverse transcription, which enabled efficient ligation of two probes complementary to the cDNA sequence on either side of the Ψ site and successful amplification in subsequent real-time quantitative PCR (qPCR), thereby achieving selective and accurate Ψ detection. Our method accurately and sensitively detected several known Ψ sites in 28S, 18S, 5.8S, and even mRNA. Moreover, pseU-TRACE could be employed to measure the Ψ fraction in RNA and explore the Ψ metabolism of different pseudouridine synthases (PUSs), providing valuable insights into the function of Ψ. Overall, pseU-TRACE represents a reliable, time-efficient and sensitive Ψ detection method.

Transcriptome-wide profiling of A-to-I RNA editing by Slic-seq.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional processing event involved in diversifying the transcriptome and is responsible for various biological processes. In this context, we developed a new method based on the highly selective cleavage activity of Endonuclease V against Inosine and the universal activity of sodium periodate against all RNAs to enrich the inosine-containing RNA and accurately identify the editing sites. We validated the reliability of our method in human brain in both Alu and non-Alu elements. The conserved sites of A-to-I editing in human cells (HEK293T, HeLa, HepG2, K562 and MCF-7) primarily occurs in the 3'UTR of the RNA, which are highly correlated with RNA binding and protein binding. Analysis of the editing sites between the human brain and mouse brain revealed that the editing of exons is more conserved than that in other regions. This method was applied to three neurological diseases (Alzheimer's, epilepsy and ageing) of mouse brain, reflecting that A-to-I editing sites significantly decreased in neuronal activity genes.

Fibroblastic reticular cell-derived exosomes are a promising therapeutic approach for septic acute kidney injury.

Sepsis-induced acute kidney injury (S-AKI) is highly lethal, and effective drugs for treatment are scarce. Previously, we reported the robust therapeutic efficacy of fibroblastic reticular cells (FRCs) in sepsis. Here, we demonstrate the ability of FRC-derived exosomes (FRC-Exos) to improve C57BL/6 mouse kidney function following cecal ligation and puncture-induced sepsis. In vivo imaging confirmed that FRC-Exos homed to injured kidneys. RNA-Seq analysis of FRC-Exo-treated primary kidney tubular cells (PKTCs) revealed that FRC-Exos influenced PKTC fate in the presence of lipopolysaccharide (LPS). FRC-Exos promoted kinase PINK1-dependent mitophagy and inhibited NLRP3 inflammasome activation in LPS-stimulated PKTCs. To dissect the mechanism underlying the protective role of Exos in S-AKI, we examined the proteins within Exos by mass spectrometry and found that CD5L was the most upregulated protein in FRC-Exos compared to macrophage-derived Exos. Recombinant CD5L treatment in vitro attenuated kidney cell swelling and surface bubble formation after LPS stimulation. FRCs were infected with a CD5L lentivirus to increase CD5L levels in FRC-Exos, which were then modified in vitro with the kidney tubular cell targeting peptide LTH, a peptide that binds to the biomarker protein kidney injury molecule-1 expressed on injured tubule cells, to enhance binding specificity. Compared with an equivalent dose of recombinant CD5L, the modified CD5L-enriched FRC-Exos selectively bound PKTCs, promoted kinase PINK-ubiquitin ligase Parkin-mediated mitophagy, inhibiting pyroptosis and improved kidney function by hindering NLRP3 inflammasome activation, thereby improving the sepsis survival rate. Thus, strategies to modify FRC-Exos could be a new avenue in developing therapeutics against kidney injury.

Detection, Clinical Application, and Manipulation of RNA Modifications.

ConspectusWith increasing research interest, more than 170 types of chemical modifications of RNA have been characterized. The epigenetic modifications of RNA do not alter the primary sequence of RNA but modulate the gene activity. Increasing numbers of regulatory functions of these RNA modifications, particularly in controlling mRNA fate and gene expression, are being discovered. To gain a deeper understanding of the biological significance and clinical prospects of RNA modifications, the development of innovative labeling and detection methodologies is of great importance. Owing to the dynamic features of RNA modifications and the fact that only a portion of genes are modified, detection methods should accurately reveal the precise distribution and modification level of RNA modifications. In general, detection methodologies identify specific RNA modifications in two ways: (1) enriching modification-containing RNAs; and (2) altering the Watson-Crick base pairing pattern to produce truncation or mutation signatures. Additionally, it is important to develop flexible and accurate manipulation tools that enable the installation or removal of RNA modifications at specific positions to investigate the biological functions of a single site. With the development of detection and manipulation methods, the scientific understanding of the biological functions of RNA modifications has increased, paving the way for applications of RNA modifications in disease diagnosis and treatments.In this Account, we provide a brief summary of recent efforts to develop methodologies for detecting RNA modifications. Through the evolution of these detection techniques, our team has uncovered the potential biological roles of RNA modifications in diseases such as diabetic cardiovascular complications, viral infections, and hematologic malignancies. We mainly summarize the recently developed strategies for manipulating RNA modifications. The advent of these programmable editing tools allows for the precise installation or removal of RNA modifications at specific positions. As a result, the biological functions of RNA modifications at these specific loci could be identified, further advancing our knowledge in this field.With this Account, we anticipate providing chemical and biological researchers with comprehensive strategies to discover the underlying mechanisms of RNA modification-mediated biological processes. Although the field of RNA modifications has undergone rapid progress in recent years, our understanding of most of these RNA modifications remains incomplete. We hope to inspire efforts to expand the toolbox for investigating RNA modifications and promote translational research on epigenetics in clinical diagnosis and treatment.

Inert Pepper aptamer-mediated endogenous mRNA recognition and imaging in living cells.

The development of RNA aptamers/fluorophores system is highly desirable for understanding the dynamic molecular biology of RNAs in vivo. Peppers-based imaging systems have been reported and applied for mRNA imaging in living cells. However, the need to insert corresponding RNA aptamer sequences into target RNAs and relatively low fluorescence signal limit its application in endogenous mRNA imaging. Herein, we remolded the original Pepper aptamer and developed a tandem array of inert Pepper (iPepper) fluorescence turn-on system. iPepper allows for efficient and selective imaging of diverse endogenous mRNA species in live cells with minimal agitation of the target mRNAs. We believe iPepper would significantly expand the applications of the aptamer/fluorophore system in endogenous mRNA imaging, and it has the potential to become a powerful tool for real-time studies in living cells and biological processing.

Antibody-Free Fluorine-Assisted Metabolic Sequencing of RNA N4-Acetylcytidine.

N4-Acetylcytidine (ac4C) has been found to affect a variety of cellular and biological processes. For a mechanistic understanding of the roles of ac4C in biology and disease, we present an antibody-free, fluorine-assisted metabolic sequencing method to detect RNA ac4C, called "FAM-seq". We successfully applied FAM-seq to profile ac4C landscapes in human 293T, HeLa, and MDA cell lines in parallel with the reported acRIP-seq method. By comparison with the classic ac4C antibody sequencing method, we found that FAM-seq is a convenient and reliable method for transcriptome-wide mapping of ac4C. Because this method holds promise for detecting nascent RNA ac4C modifications, we further investigated the role of ac4C in regulating chemotherapy drug resistance in chronic myeloid leukemia. The results indicated that drug development or combination therapy could be enhanced by appreciating the key role of ac4C modification in cancer therapy.

Keth-seq for transcriptome-wide RNA structure mapping.

RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.

Orthogonal Demethylase-Activated Deoxyribozyme for Intracellular Imaging and Gene Regulation.

The epigenetic modification of nucleic acids represents a versatile approach for achieving high-efficient control over gene expression and transcription and could dramatically expand their biosensing and therapeutic applications. Demethylase-involved removal of N6-methyladenine (m6A) represents one of the vital epigenetic reprogramming events, yet its direct intracellular evaluation and as-guided gene regulation are extremely rare. The endonuclease-mimicking deoxyribozyme (DNAzyme) is a catalytically active DNA that enables the site-specific cleavage of the RNA substrate, and several strategies have imparted the magnificent responsiveness to DNAzyme by using chemical and light stimuli. However, the epigenetic regulation of DNAzyme has remained largely unexplored, leaving a significant gap in responsive DNA nanotechnology. Herein, we reported an epigenetically responsive DNAzyme system through the in vitro selection of an exquisite m6A-caged DNAzyme that could be specifically activated by FTO (fat mass and obesity-associated protein) demethylation for precise intracellular imaging-directed gene regulation. Based on a systematic investigation, the active DNAzyme configuration was potently disrupted by the site-specific incorporation of m6A modification and subsequently restored into the intact DNAzyme structure via the tunable FTO-specific removal of m6A-caging groups under a variety of conditions. This orthogonal demethylase-activated DNAzyme amplifier enables the robust and accurate monitoring of FTO and its inhibitors in live cells. Moreover, the simple demethylase-activated DNAzyme facilitates the assembly of an intelligent self-adaptive gene regulation platform for knocking down demethylase with the ultimate apoptosis of tumor cells. As a straightforward and scarless m6A removal strategy, the demethylase-activated DNAzyme system offers a versatile toolbox for programmable gene regulation in synthetic biology.

N3 -Kethoxal-Based Bioorthogonal Intracellular RNA Labeling.

There is growing interest in developing intracellular RNA tools. Herein, we describe a strategy for N3 -kethoxal (N3 K)-based bioorthogonal intracellular RNA functionalization. With N3 K labeling followed by an in vivo click reaction with DBCO derivatives, RNA can be modified with fluorescent or phenol groups. This strategy provides a new way of labeling RNA inside cells.

4-Thiouridine-Enhanced Peroxidase-Generated Biotinylation of RNA.

Peroxidase-generated proximity labeling is in widespread use to study subcellular proteomes and the protein interaction networks in living cells, but the development of subcellular RNA labeling is limited. APEX-seq has emerged as a new method to study subcellular RNA in living cells, but the labeling of RNA still has room to improve. In this work, we describe 4-thiouridine (s4 U)-enhanced peroxidase-generated biotinylation of RNA with high efficiency. The incorporation of s4 U could introduce additional sites for RNA labeling, enhanced biotinylation was observed on monomer, model oligo RNA and total RNA. Through the s4 U metabolic approach, the in vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit RNA isolation and study for the spatial transcriptome.

Highly sensitive detection of 6mA at single-base resolution based on A-C mismatch.

We first demonstrated that 6mA can be selectively recognized based on the selective ligation reaction of DNA ligase toward A-C mismatch and 6mA-C mismatch. This method, when further combined with amplification using RCA, achieved highly sensitive identification of 6mA in dsDNA at single-base resolution.

A m6 A Sensing Method by Its Impact on the Stability of RNA Double Helix.

N6 -Methyladenosine (m6 A) is one of the most important RNA modifications in epigenetics. The development of detection method for m6 A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m6 A modification and target RNAs without m6 A modification. We verified the fact that the presence of m6 A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m6 A detection methods in the future.

A sensitive and radiolabeling-free method for pseudouridine detection.

Existing methodologies for detecting Pseudouridine (Ψ) mostly use CMCT labeling or radiolabeling. Described herein is a sensitive and quantitative method for Ψ detection that does not need this labelling. This approach combines the selectivity of a 10-23 DNAzyme, which can distinguish Ψ from uridine (U), with rolling circle amplification (RCA) to increase the sensitivity of the assay.

Biotinylation and isolation of an RNA G-quadruplex based on its peroxidase-mimicking activity.

RNA G-quadruplexes (rG4s) are important RNA secondary structures considering their significance in regulating numerous cellular processes. Described herein is an rG4 detecting and isolation method, which exploits the complex of rG4 and hemin to mimic peroxidase. In the presence of biotin tyramide and hydrogen peroxide, rG4s can be selectively self-biotinylated and easily isolated from a complex RNA mixture using streptavidin magnetic beads.

Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution.

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.

Luminescence Sensing for Qualitative and Quantitative Detection of 5-Methylcytosine.

5-Methylcytosine (5mC) is revealed as a heritable epigenetic modification in genomic DNA. It has been reported that cytosine/guanine dinucleotides (CpG) hypermethylation in the promoter regions of tumor suppressor genes is related with inappropriate gene silencing, so the determination of 5mC in the CpG islands of mammals has attracted much attention. In this paper, a luminescence sensing strategy based on bisulfite treatment, asymmetric polymerase chain reaction (PCR), and adenosine triphosphate (ATP)-releasing nucleotide is proposed. With little background, this method can provide accurate quantitative information about methylation changes at CpG sites, even at a specific site. The proposed method can be successfully employed to determine the methylation status of three hepatocellular carcinomas (HCC) related genes in clinical tissues.

Existence of Diverse Modifications in Small-RNA Species Composed of 16-28†Nucleotides.

RNA contains diverse modifications that exert an important influence in a variety of cellular processes. So far, more than 150 modifications have been identified in various RNA species, mainly in ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). In contrast to rRNA, tRNA, and mRNA, the known modifications in small RNA species have been primarily limited to 2'-O-ribose methylation in plants and inosine in mammals. The methylation of small RNAs in mammals is still unclear. Current methods widely used in the characterization of small RNAs are mainly based on the strategy of nucleic acid hybridization and sequencing, which cannot characterize modifications in small RNAs. Herein, we have systematically investigated modifications in small RNAs composed of 16-28 nucleotides (nt) by establishing an effective isolation and neutral enzymatic digestion of small RNAs in combination with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This method allowed us to simultaneously detect 57 different types of nucleoside modification. By using this approach, we revealed 24 modifications in small RNAs comprising 16-28 nt from human cells. In addition, we found that the obesity-associated protein (FTO) may demethylate N6 -methyladenosine (m6 A) and N6 ,2'-O-dimethyladenosine (m6 Am) in small RNAs of 16-28 nt. Our study demonstrates the existence of diverse modifications in small RNAs composed of 16-28 nt, which may promote in-depth understanding of the regulatory roles of noncoding RNAs.