High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants.

Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles in OsALS and creating synonymous mutations in OsSPL14 to resist OsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox.

Directed-evolution mutations enhance DNA-binding affinity and protein stability of the adenine base editor ABE8e.

Adenine base editors (ABEs), consisting of CRISPR Cas nickase and deaminase, can chemically convert the A:T base pair to G:C. ABE8e, an evolved variant of the base editor ABE7.10, contains eight directed evolution mutations in its deaminase TadA8e that significantly increase its base editing activity. However, the functional implications of these mutations remain unclear. Here, we combined molecular dynamics (MD) simulations and experimental measurements to investigate the role of the directed-evolution mutations in the base editing catalysis. MD simulations showed that the DNA-binding affinity of TadA8e is higher than that of the original deaminase TadA7.10 in ABE7.10 and is mainly driven by electrostatic interactions. The directed-evolution mutations increase the positive charge density in the DNA-binding region, thereby enhancing the electrostatic attraction of TadA8e to DNA. We identified R111, N119 and N167 as the key mutations for the enhanced DNA binding and confirmed them by microscale thermophoresis (MST) and in vivo reversion mutation experiments. Unexpectedly, we also found that the directed mutations improved the thermal stability of TadA8e by ~ 12 °C (Tm, melting temperature) and that of ABE8e by ~ 9 °C, respectively. Our results demonstrate that the directed-evolution mutations improve the substrate-binding ability and protein stability of ABE8e, thus providing a rational basis for further editing optimisation of the system.

Insights on multi-spectroscopic approaches for estimating the in vitro binding of favipiravir with adenine nucleotide.

Favipiravir (FVP) is an oral antiviral drug approved in 2021 for the treatment of COVID-19. It is a pyrazine derivative that can be integrated into anti-viral RNA products to inhibit viral replication. While, adenine is a purine nucleobase that is found in deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to generate genetic information. For the first time, the binding mechanism between FVP and adenine was determined using different techniques, including UV-visible spectrophotometry, spectrofluorimetry, synchronous fluorescence (SF) spectroscopy, Fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET), and metal ion complexation. The fluorescence spectra indicated that FVP is bound to adenine via Van der Waals forces and hydrogen bonding through a spontaneous binding process (ΔGο < 0). The quenching mechanism was found to be static. Various temperature settings were used to investigate thermodynamic characteristics, such as binding forces, binding constants, and the number of binding sites. The reaction parameters, including the enthalpy change (ΔHο) and entropy change (ΔSο), were calculated using Van't Hoff's equation. The findings demonstrated that the adenine-FVP binding was endothermic. Furthermore, the results of the experiments revealed that some metal ions (K+, Ca+2, Co+2, Cu+2, and Al+3) might facilitate the binding interaction between FVP and adenine. Slight changes are observed in the FTIR spectra of adenine, indicating the binding interaction between adenine and FVP. This study may be useful in understanding the pharmacokinetic characteristics of FVP and how the drug binds to adenine to prevent any side effects.

Structural basis for substrate recognition by a S-adenosylhomocysteine hydrolase Lpg2021 from Legionella pneumophila.

S-Adenosyl-l-homocysteine hydrolase (SAHH) is a crucial enzyme that governs S-adenosyl methionine (SAM)-dependent methylation reactions within cells and regulates the intracellular concentration of SAH. Legionella pneumophila, the causative pathogen of Legionnaires' disease, encodes Lpg2021, which is the first identified dimeric SAHH in bacteria and is a promising target for drug development. Here, we report the structure of Lpg2021 in its ligand-free state and in complexes with adenine (ADE), adenosine (ADO), and 3-Deazaneplanocin A (DZNep). X-ray crystallography, isothermal titration calorimetry (ITC), and molecular docking were used to elucidate the binding mechanisms of Lpg2021 to its substrates and inhibitors. Virtual screening was performed to identify potential Lpg2021 inhibitors. This study contributes a novel perspective to the understanding of SAHH evolution and establishes a structural framework for designing specific inhibitors targeting pathogenic Legionella pneumophila SAHH.

Semiconservative transmission of DNA N 6-adenine methylation in a unicellular eukaryote.

Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

Isomerization Pathways of a Mismatched Base Pair of A:8OG in Free Duplex DNA.

The A:8OG base pair (bp) is the outcome of DNA replication of the mismatched C:8OG bp. A high A:8OG bp population increases the C/G to A/T transversion mutation, which is responsible for various diseases. MutY is an important enzyme in the error-proof cycle and reverts A:8OG to C:8OG bp by cleaving adenine from the A:8OG bp. Several X-ray crystallography studies have determined the structure of MutY during the lesion scanning and lesion recognition stages. Interestingly, glycosidic bond (χ) angles of A:8OG bp in those two lesion recognition structures were found to differ, which implies that χ-torsion isomerization should occur during the lesion recognition process. In this study, as a first step to understanding this isomerization process, we characterized the intrinsic dynamic features of A:8OG in free DNAs by a free energy landscape simulation at the all-atom level. In this study, four isomerization states were assigned in the order of abundance: Aanti:8OGsyn > Aanti:8OGanti > Asyn:8OGanti ≈ Asyn:8OGsyn. Of these bp states, only 8OG in Asyn:8OGanti was located in the extrahelical space, whereas the purine bases (A and 8OG) in the other bp states remained inside the DNA helix. Also, free energy landscapes showed that the isomerization processes connecting these four bp states proceeded mostly in the intrahelical space via successive single glycosidic bond rotations of either A or 8OG.

Base editing correction of OCRL in Lowe syndrome: ABE-mediated functional rescue in patient-derived fibroblasts.

Lowe syndrome, a rare X-linked multisystem disorder presenting with major abnormalities in the eyes, kidneys, and central nervous system, is caused by mutations in OCRL gene (NG_008638.1). Encoding an inositol polyphosphate 5-phosphatase, OCRL catalyzes the hydrolysis of PI(4,5)P2 into PI4P. There are no effective targeted treatments for Lowe syndrome. Here, we demonstrate a novel gene therapy for Lowe syndrome in patient fibroblasts using an adenine base editor (ABE) that can efficiently correct pathogenic point mutations. We show that ABE8e-NG-based correction of a disease-causing mutation in a Lowe patient-derived fibroblast line containing R844X mutation in OCRL gene, restores OCRL expression at mRNA and protein levels. It also restores cellular abnormalities that are hallmarks of OCRL dysfunction, including defects in ciliogenesis, microtubule anchoring, α-actinin distribution, and F-actin network. The study indicates that ABE-mediated gene therapy is a feasible treatment for Lowe syndrome, laying the foundation for therapeutic application of ABE in the currently incurable disease.

m6A RNA methylation: The latent string-puller in fibrosis.

Fibrosis is a pathological phenomenon characterized by the aberrant accumulation of extracellular matrix (ECM) in tissues. Fibrosis is a universally age-related disease involving that many organs and is the final stage of many chronic inflammatory diseases, which often threaten the patient's health. Undoubtedly, fibrosis has become a serious economic and health burden worldwide, However, the pathogenesis of fibrosis is complex. Further, the key molecules still remain to be unraveled. Hence, so far, there have been no effective treatments designed against the key targets of fibrosis. The methylation modification on the nitrogen atom at position 6 of adenine (m6A) is the most common mRNA modification in mammals. There is increasing evidence that m6A is actively involved in the pathogenesis of fibrosis. This review aims to highlight m6A-associated mechanisms and functions in several organic fibrosis, which implies that m6A is universal and critical for fibrosis and summarize the outlook of m6A in the treatment of fibrosis. This may light up the unknown aspects of this condition for researchers interested to explore fibrosis further.

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Bacterial adenine cross-feeding stems from a purine salvage bottleneck.

Diverse ecosystems host microbial relationships that are stabilized by nutrient cross-feeding. Cross-feeding can involve metabolites that should hold value for the producer. Externalization of such communally valuable metabolites is often unexpected and difficult to predict. Previously, we discovered purine externalization by Rhodopseudomonas palustris by its ability to rescue an Escherichia coli purine auxotroph. Here we found that an E. coli purine auxotroph can stably coexist with R. palustris due to purine cross-feeding. We identified the cross-fed purine as adenine. Adenine was externalized by R. palustris under diverse growth conditions. Computational modeling suggested that adenine externalization occurs via diffusion across the cytoplasmic membrane. RNAseq analysis led us to hypothesize that adenine accumulation and externalization stem from a salvage pathway bottleneck at the enzyme encoded by apt. Ectopic expression of apt eliminated adenine externalization, supporting our hypothesis. A comparison of 49 R. palustris strains suggested that purine externalization is relatively common, with 16 strains exhibiting the trait. Purine externalization was correlated with the genomic orientation of apt, but apt orientation alone could not always explain purine externalization. Our results provide a mechanistic understanding of how a communally valuable metabolite can participate in cross-feeding. Our findings also highlight the challenge in identifying genetic signatures for metabolite externalization.

Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Interaction-based screening, Monte Carlo Bayesian inference-based de novo design and in vitro verification of adenine-binding peptide.

One of the main reasons for hyperuricemia is high purine intake. The primary strategy for treating hyperuricemia is blocking the purine metabolism enzyme. However, by binding the purine bases directly, we suggested a unique therapeutic strategy that might interfere with purine metabolism. There have been numerous reports of extensive interactions between proteins and purine bases. Adenine, constituting numerous protein co-factors, can interact with the adenine-binding motif. Using Bayesian Inference and Markov chain Monte Carlo sampling, we created a novel adenine-binding peptide Ile-Tyr-Val-Thr based on the structure of the adenine-binding motifs. Ile-Tyr-Val-Thr generates a semi-pocket that can clip the adenine within, as demonstrated by docking. Then, using thermodynamic techniques, the interaction between Ile-Tyr-Val-Thr and adenine was confirmed. The KD value is 1.50e-5 (ΔH = -20.2 kJ/mol and ΔG = -27.6 kJ/mol), indicating the high affinity. In brief, the adenine-binding peptide Ile-Tyr-Val-Thr may help lower uric acid level by blocking the absorption of food-derived adenine.

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation.

KEY MESSAGE: Barley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5' terminal nucleotide of small RNAs, demonstrating functional conservation and divergence. The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5' adenine residue, while also accepting 5' guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5' adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

Recovery of Water Homeostasis in Adenine-Induced Kidney Disease Is Mediated by Increased AQP2 Membrane Targeting.

Chronic kidney disease (CKD) represents a major public health burden with increasing prevalence. Current therapies focus on delaying CKD progression, underscoring the need for innovative treatments. This necessitates animal models that accurately reflect human kidney pathologies, particularly for studying potential reversibility and regenerative mechanisms, which are often hindered by the progressive and irreversible nature of most CKD models. In this study, CKD was induced in mice using a 0.2% adenine-enriched diet for 4 weeks, followed by a recovery period of 1 or 2 weeks. The aim was to characterize the impact of adenine feeding on kidney function and injury as well as water and salt homeostasis throughout disease progression and recovery. The adenine diet induced CKD is characterized by impaired renal function, tubular injury, inflammation, and fibrosis. A significant decrease in urine osmolality, coupled with diminished aquaporin-2 (AQP2) expression and membrane targeting, was observed after adenine treatment. Intriguingly, these parameters exhibited a substantial increase after a two-week recovery period. Despite these functional improvements, only partial reversal of inflammation, tubular damage, and fibrosis were observed after the recovery period, indicating that the inclusion of the molecular and structural parameters is needed for a more complete monitoring of kidney status.

Identification of AtALKBH1A and AtALKBH1D as DNA N6-adenine demethylases in Arabidopsis thaliana.

DNA N6-methyladenine (6 mA) has recently been discovered as a novel DNA modification in animals and plants. In mammals, AlkB homolog 1 (ALKBH1) has been identified as a DNA 6 mA demethylase. ALKBH1 tightly controls the DNA 6 mA methylation level of mammalian genomes and plays important role in regulating gene expression. DNA 6 mA methylation has also been reported to exist in plant genomes, however, the plant DNA 6 mA demethylases and their function remain largely unknown. Here we identify homologs of ALKBH1 as DNA 6 mA demethylases in Arabidopsis. We discover that there are four homologs of ALKBH1, AtALKBH1A, AtALKBH1B, AtALKBH1C and AtALKBH1D, in Arabidopsis. In vitro enzymatic activity studies reveal that AtALKBH1A and 1D can efficiently erase DNA 6 mA methylation. Loss of function of AtALKBH1A and AtALKBH1D causes elevated DNA 6 mA methylation levels in vivo. atalkbh1a/1d mutant displays delayed seed gemination. Based on our RNA-seq data, we find some regulators of seed gemination are dysregulated in atalkbh1a/1d, and the dysregulation is correlated with changes of DNA 6 mA methylation levels. This study identifies plant DNA 6 mA demethylases and reports the function of DNA 6 mA methylation in regulating seed germination.

Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope.

Nme2Cas9 has been established as a genome editing platform with compact size, high accuracy, and broad targeting range, including single-AAV-deliverable adenine base editors. Here, we engineer Nme2Cas9 to further increase the activity and targeting scope of compact Nme2Cas9 base editors. We first use domain insertion to position the deaminase domain nearer the displaced DNA strand in the target-bound complex. These domain-inlaid Nme2Cas9 variants exhibit shifted editing windows and increased activity in comparison to the N-terminally fused Nme2-ABE. We next expand the editing scope by swapping the Nme2Cas9 PAM-interacting domain with that of SmuCas9, which we had previously defined as recognizing a single-cytidine PAM. We then use these enhancements to introduce therapeutically relevant edits in a variety of cell types. Finally, we validate domain-inlaid Nme2-ABEs for single-AAV delivery in vivo.

Optimization and validation of a UPLC-MS/MS assay for simultaneous quantification of 2,8-dihydroxyadenine, adenine, allopurinol, oxypurinol and febuxostat in human plasma.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare , hereditary disorder characterized by renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stone formation and chronic kidney disease (CKD). Treatment with a xanthine oxidoreductase inhibitor, allopurinol or febuxostat, reduces urinary DHA excretion and slows the progression of CKD. The method currently used for therapeutic monitoring of APRT deficiency lacks specificity and thus, a more reliable measurement technique is needed. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma was optimized and validated. Plasma samples were prepared with protein precipitation using acetonitrile followed by evaporation. The chemometric approach design of experiments was implemented to optimize gradient steepness, amount of organic solvent, flow rate, column temperature, cone voltage, desolvation temperature and desolvation flow rate. Experimental screening was conducted using fractional factorial design with addition of complementary experiments at the axial points for optimization of peak area, peak resolution and peak width. The assay was validated according to the US Food and Drug Administration guidelines for bioanalytical method validation over the concentration range of 50 to 5000 ng/mL for DHA, allopurinol and febuxostat, 100 to 5000 ng/mL for adenine and 50 to 12,000 ng/mL for oxypurinol, with r2 ≥ 0.99. The analytical assay achieved acceptable performance of accuracy (-10.8 to 8.3 %) and precision (CV < 15 %). DHA, adenine, allopurinol, oxypurinol and febuxostat were stable in plasma samples after five freeze-thaw cycles at -80 °C and after storage at -80 °C for 12 months. The assay was evaluated for quantification of the five analytes in clinical plasma samples from six APRT deficiency patients and proved to be both efficient and accurate. The proposed assay will be valuable for guiding pharmacotherapy and thereby contribute to improved and more personalized care for patients with APRT deficiency.

Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.

Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis.

Epigenetic marks on histones and DNA, such as DNA methylation at N6-adenine (6mA), play crucial roles in gene expression and genome maintenance, but their deposition and function in microalgae remain largely uncharacterized. Here, we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing. Found in 0.1% of adenines, 6mA sites are mostly enriched at the AGGYV motif, more abundant in transposons and 3' untranslated regions, and associated with active transcription. Moreover, 6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcription termination sites. Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes, indicating a positive interaction between 6mA and general transcription factors. Furthermore, knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor, sulfate transporter, glycosyl transferase, and lipase genes that underlie reductions in biomass and oil productivity. By contrast, knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth. Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.

Role of RNA Modifications, Especially m6A, in Aflatoxin Biosynthesis of Aspergillus flavus.

RNA modifications play key roles in eukaryotes, but the functions in Aspergillus flavus are still unknown. Temperature has been reported previously to be a critical environmental factor that regulates the aflatoxin production of A. flavus, but much remains to be learned about the molecular networks. Here, we demonstrated that 12 kinds of RNA modifications in A. flavus were significantly changed under 29 °C compared to 37 °C incubation; among them, m6A was further verified by a colorimetric method. Then, the transcriptome-wide m6A methylome and m6A-altered genes were comprehensively illuminated through methylated RNA immunoprecipitation sequencing and RNA sequencing, from which 22 differentially methylated and expressed transcripts under 29 °C were screened out. It is especially notable that AFCA_009549, an aflatoxin biosynthetic pathway gene (aflQ), and the m6A methylation of its 332nd adenine in the mRNA significantly affect aflatoxin biosynthesis in A. flavus both on media and crop kernels. The content of sterigmatocystin in both ΔaflQ and aflQA332C strains was significantly higher than that in the WT strain. Together, these findings reveal that RNA modifications are associated with secondary metabolite biosynthesis of A. flavus.