Antibiotic-Induced Gut Microbiota Dysbiosis Modulates Host Transcriptome and m6A Epitranscriptome via Bile Acid Metabolism.

Gut microbiota can influence host gene expression and physiology through metabolites. Besides, the presence or absence of gut microbiome can reprogram host transcriptome and epitranscriptome as represented by N6-methyladenosine (m6A), the most abundant mammalian mRNA modification. However, which and how gut microbiota-derived metabolites reprogram host transcriptome and m6A epitranscriptome remain poorly understood. Here, investigation is conducted into how gut microbiota-derived metabolites impact host transcriptome and m6A epitranscriptome using multiple mouse models and multi-omics approaches. Various antibiotics-induced dysbiotic mice are established, followed by fecal microbiota transplantation (FMT) into germ-free mice, and the results show that bile acid metabolism is significantly altered along with the abundance change in bile acid-producing microbiota. Unbalanced gut microbiota and bile acids drastically change the host transcriptome and the m6A epitranscriptome in multiple tissues. Mechanistically, the expression of m6A writer proteins is regulated in animals treated with antibiotics and in cultured cells treated with bile acids, indicating a direct link between bile acid metabolism and m6A biology. Collectively, these results demonstrate that antibiotic-induced gut dysbiosis regulates the landscape of host transcriptome and m6A epitranscriptome via bile acid metabolism pathway. This work provides novel insights into the interplay between microbial metabolites and host gene expression.

A KDPG sensor RccR governs Pseudomonas aeruginosa carbon metabolism and aminoglycoside antibiotic tolerance.

Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The extraordinary capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residue R152, K270 or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG-sensing mechanism by RccR.

Co-effects of m6A and chromatin accessibility dynamics in the regulation of cardiomyocyte differentiation.

BACKGROUND: Cardiomyocyte growth and differentiation rely on precise gene expression regulation, with epigenetic modifications emerging as key players in this intricate process. Among these modifications, N6-methyladenosine (m6A) stands out as one of the most prevalent modifications on mRNA, exerting influence over mRNA metabolism and gene expression. However, the specific function of m6A in cardiomyocyte differentiation remains poorly understood. RESULTS: We investigated the relationship between m6A modification and cardiomyocyte differentiation by conducting a comprehensive profiling of m6A dynamics during the transition from pluripotent stem cells to cardiomyocytes. Our findings reveal that while the overall m6A modification level remains relatively stable, the m6A levels of individual genes undergo significant changes throughout cardiomyocyte differentiation. We discovered the correlation between alterations in chromatin accessibility and the binding capabilities of m6A writers, erasers, and readers. The changes in chromatin accessibility influence the recruitment and activity of m6A regulatory proteins, thereby impacting the levels of m6A modification on specific mRNA transcripts. CONCLUSION: Our data demonstrate that the coordinated dynamics of m6A modification and chromatin accessibility are prominent during the cardiomyocyte differentiation.

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.

Mapping single-nucleotide m6A by m6A-REF-seq.

The past few years have witnessed rapid progress in the field of RNA modifications. As the most prevailing modification on eukaryotic mRNA, m6A is characterized to play a vital role in various cellular activities. However, limitations of the detection method impede functional studies of m6A. Here we introduce m6A-REF-seq, a powerful and straightforward method to identify m6A at single-nucleotide resolution. m6A-REF-seq relies on the recognition of RNA endonuclease MazF towards m6A at the ACA motif, providing an orthogonal method independent of the m6A antibody being adopted by most of current methods. We describe a detailed protocol to perform m6A-REF-seq, including NGS library construction and sequencing data analysis. In particular, we describe an optimized assay to validate individual m6A sites identified by m6A-REF-seq, which can also be applied to detect any candidate m6A sites.

Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library.

Recent years have witnessed rapid progress in the field of epitranscriptomics. Functional interpretation of the epitranscriptome relies on sequencing technologies that determine the location and stoichiometry of various RNA modifications. However, contradictory results have been reported among studies, bringing the biological impacts of certain RNA modifications into doubt. Here, we develop a synthetic RNA library resembling the endogenous transcriptome but without any RNA modification. By incorporating this modification-free RNA library into established mapping techniques as a negative control, we reveal abundant false positives resulting from sequence bias or RNA structure. After calibration, precise and quantitative mapping expands the understanding of two representative modification types, N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We propose that this approach provides a systematic solution for the calibration of various RNA-modification mappings and holds great promise in epitranscriptomic studies.

Targeted genetic screening in bacteria with a Cas12k-guided transposase.

Microbes employ sophisticated cellular networks encoded by complex genomes to rapidly adapt to changing environments. High-throughput genome engineering methods are valuable tools for functionally profiling genotype-phenotype relationships and understanding the complexity of cellular networks. However, current methods either rely on special homologous recombination systems and are thus applicable in only limited bacterial species or can generate only nonspecific mutations and thus require extensive subsequent screening. Here, we report a site-specific transposon-assisted genome engineering (STAGE) method that allows high-throughput Cas12k-guided mutagenesis in various microorganisms, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Exploiting the powerful STAGE technique, we construct a site-specific transposon mutant library that focuses on all possible transcription factors (TFs) in P. aeruginosa, enabling the comprehensive identification of essential genes and antibiotic-resistance-related factors. Given its broad host range activity and easy programmability, this method can be widely adapted to diverse microbial species for rapid genome engineering and strain evolution.

Targeted RNA N6 -Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells.

Deficiency of the N6 -methyladenosine (m6 A) methyltransferase complex results in global reduction of m6 A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6 A methylation affects cell fate decisions due to the inability to modulate individual m6 A modification in ESCs with precise temporal control. Here, a targeted RNA m6 A erasure (TRME) system is developed to achieve site-specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline-inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6 A site of mRNA and increase mRNA stability with limited off-target effects. It is further demonstrated that temporal m6 A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6 A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.

The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.

The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

RNA m6A Modification Functions in Larval Development and Caste Differentiation in Honeybee (Apis mellifera).

Genetically identical female honeybee larvae with different diets develop into sterile workers or fertile queens. It remains unknown whether the reversible RNA N6-methyladenosine (m6A) mark functionally impact this "caste differentiation." Here, we profile the transcriptome-wide m6A methylome of honeybee queen and worker larvae at three instar stages and discover that m6A methylation dynamics are altered by differential feeding. Multiple methylome comparisons show an obvious increase in m6A marks during larval development and reveal a negative correlation between gene expression and m6A methylation. Notably, we find that worker larvae contain more hypermethylated m6A peaks than do queen larvae, and many caste-differentiation-related transcripts are differentially methylated. Chemical suppression of m6A methylation in worker larvae by 3-deazaadenosine (DAA) reduces overall m6A methylation levels and triggers worker larvae to develop queen caste features. Thus, our study demonstrates that m6A functionally impacts caste differentiation and larval development, yet it does not exclude potential contributions from other factors.

Crystal structure of the yeast heterodimeric ADAT2/3 deaminase.

BACKGROUND: The adenosine-to-inosine (A-to-I) editing in anticodons of tRNAs is critical for wobble base-pairing during translation. This modification is produced via deamination on A34 and catalyzed by the adenosine deaminase acting on tRNA (ADAT) enzyme. Eukaryotic ADATs are heterodimers composed of the catalytic subunit ADAT2 and the structural subunit ADAT3, but their molecular assemblies and catalytic mechanisms are largely unclear. RESULTS: Here, we report a 2.8-Å crystal structure of Saccharomyces cerevisiae ADAT2/3 (ScADAT2/3), revealing its heterodimeric assembly and substrate recognition mechanism. While each subunit clearly contains a domain resembling their prokaryotic homolog TadA, suggesting an evolutionary gene duplication event, they also display accessory domains for additional structural or functional purposes. The N-lobe of ScADAT3 exhibits a positively charged region with a potential role in the recognition and binding of tRNA, supported by our biochemical analysis. Interestingly, ScADAT3 employs its C-terminus to block tRNA's entry into its pseudo-active site and thus inactivates itself for deamination despite the preservation of a zinc-binding site, a mechanism possibly shared only among yeasts. CONCLUSIONS: Combining the structural with biochemical, bioinformatic, and in vivo functional studies, we propose a stepwise model for the pathway of deamination by ADAT2/3. Our work provides insight into the molecular mechanism of the A-to-I editing by the eukaryotic ADAT heterodimer, especially the role of ADAT3 in catalysis.

Mapping and editing of nucleic acid modifications.

Modification on nucleic acid plays a pivotal role in controlling gene expression. Various kinds of modifications greatly increase the information-encoding capacity of DNA and RNA by introducing extra chemical group to existing bases instead of altering the genetic sequences. As a marker on DNA or RNA, nucleic acid modification can be recognized by specific proteins, leading to versatile regulation of gene expression. However, modified and regular bases are often indistinguishable by most conventional molecular methods, impeding detailed functional studies that require the information of genomic location. Recently, new technologies are emerging to resolve the positions of varied modifications on both DNA and RNA. Intriguingly, by integrating regional targeting tools and effector proteins, researchers begin to actively control the modification status of desired gene in vivo. In this review, we summarize the characteristics of DNA and RNA modifications, the available mapping and editing tools, and the potential application as well as deficiency of these technologies in basic and translational researches.

Single-base mapping of m6A by an antibody-independent method.

N 6-methyladenosine (m6A) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput m6A identification method depends on the anti-m6A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of m6A. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic, single-base m6A maps generated by m6A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual m6A sites, confirming the high reliability and accuracy of m6A-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that m6A sites are conserved with single-nucleotide specificity and tend to cluster among species.

N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA.

BACKGROUND: N6-methyldeoxyadenosine (6mA or m6dA) was shown more than 40 years ago in simple eukaryotes. Recent studies revealed the presence of 6mA in more prevalent eukaryotes, even in vertebrates. However, functional characterizations have been limited. RESULTS: We use Tetrahymena thermophila as a model organism to examine the effects of 6mA on nucleosome positioning. Independent methods reveal the enrichment of 6mA near and after transcription start sites with a periodic pattern and anti-correlation relationship with the positions of nucleosomes. The distribution pattern can be recapitulated by in vitro nucleosome assembly on native Tetrahymena genomic DNA but not on DNA without 6mA. Model DNA containing artificially installed 6mA resists nucleosome assembling compared to unmodified DNA in vitro. Computational simulation indicates that 6mA increases dsDNA rigidity, which disfavors nucleosome wrapping. Knockout of a potential 6mA methyltransferase leads to a transcriptome-wide change of gene expression. CONCLUSIONS: These findings uncover a mechanism by which DNA 6mA assists to shape the nucleosome positioning and potentially affects gene expression.

YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs.

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective 'reader' proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

DNA N6-methyladenine in metazoans: functional epigenetic mark or bystander?

The DNA-adenine modification N6-methyladenine (6mA), initially thought to be mainly restricted to prokaryotes and certain unicellular eukaryotes, has recently been found in metazoans. Proposed functions vary from gene activation to transposon suppression. However, since most metazoan genomes possess 5-methylcytosine (5mC) as a dominant epigenetic mark, it raises the question of why 6mA is required. This Perspective summarizes the latest discoveries and suggests potential functional roles for 6mA in metazoan genomes.