m3C32 tRNA modification controls serine codon-biased mRNA translation, cell cycle, and DNA-damage response.

The epitranscriptome includes a diversity of RNA modifications that influence gene expression. N3-methylcytidine (m3C) mainly occurs in the anticodon loop (position C32) of certain tRNAs yet its role is poorly understood. Here, using HAC-Seq, we report comprehensive METTL2A/2B-, METTL6-, and METTL2A/2B/6-dependent m3C profiles in human cells. METTL2A/2B modifies tRNA-arginine and tRNA-threonine members, whereas METTL6 modifies the tRNA-serine family. However, decreased m3C32 on tRNA-Ser-GCT isodecoders is only observed with combined METTL2A/2B/6 deletion. Ribo-Seq reveals altered translation of genes related to cell cycle and DNA repair pathways in METTL2A/2B/6-deficient cells, and these mRNAs are enriched in AGU codons that require tRNA-Ser-GCT for translation. These results, supported by reporter assays, help explain the observed altered cell cycle, slowed proliferation, and increased cisplatin sensitivity phenotypes of METTL2A/2B/6-deficient cells. Thus, we define METTL2A/2B/6-dependent methylomes and uncover a particular requirement of m3C32 tRNA modification for serine codon-biased mRNA translation of cell cycle, and DNA repair genes.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production.

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

Improving the level of the cytidine biosynthesis in E. coli through atmospheric room temperature plasma mutagenesis and metabolic engineering.

AIMS: Cytidine, as an important commercial precursor in the chemical synthesis of antiviral and antitumor drugs, is in great demand in the market. Therefore, the purpose of this study is to build a microbial cell factory with high cytidine production. METHODS AND RESULTS: A mutant E. coli NXBG-11-F34 with high tolerance to uridine monophosphate structural analogs and good genetic stability was obtained by atmospheric room temperature plasma (ARTP) mutagenesis combined with high-throughput screening. Then, the udk and rihA genes involved in cytidine catabolism were knocked out by CRISPR/Cas9 gene editing technology, and the recombinant strain E. coli NXBG-13 was constructed. The titer, yield, and productivity of cytidine fermented in a 5 l bioreactor were 15.7 g l-1, 0.164 g g-1, and 0.327 g l-1 h-1, respectively. Transcriptome analysis of the original strain and the recombinant strain E. coli NXBG-13 showed that the gene expression profiles of the two strains changed significantly, and the cytidine de novo pathway gene of the recombinant strain was up-regulated significantly. CONCLUSIONS: ARTP mutagenesis combined with metabolic engineering is an effective method to construct cytidine-producing strains.

Impacts of in-vitro zebularine treatment on genome-wide DNA methylation and transcriptomic profiles in Salix purpurea L.

Renewable energy resources such as biomass are crucial for a sustainable global society. Trees are a major source of lignocellulosic biomass, which can vary in response to different environmental factors owing to epigenetic regulation, such as DNA C-methylation. To investigate the effects of DNA methylation on plant development and wood formation, and its impacts on gene expression, with a focus on secondary cell wall (SCW)-associated genes, Salix purpurea plantlets were cloned from buds derived from a single hybrid tree for both treatment and control conditions. For the treatment condition, buds were exposed to 50 µM zebularine in vitro and a combined strategy of whole-genome bisulfite sequencing (WGBS) and RNA-seq was employed to examine the methylome and transcriptome profiles of different tissues collected at various time points under both conditions. Transcriptomic and methylome data revealed that most of the promoter and gene body demethylation had no marked effects on the expression profiles of genes. Nevertheless, gene expression tended to decrease with the increased methylation levels of genes with highly methylated promoters. Results indicated that demethylation is less evident in centromeric regions and sex chromosomes. Promoters of secondary cell wall-associated genes, such as 4-coumarate-CoA ligase-like and Rac-like GTP-binding protein RHO, were differentially methylated in the secondary xylem samples collected from two-month potted treated plants compared to control samples. Our results provide novel insights into DNA methylation and gene expression landscapes and a basis for investigating the epigenetic regulation of wood formation in S. purpurea as a model plant for bioenergy species.

NAT10 and cytidine acetylation in mRNA: intersecting paths in development and disease.

N4-acetylcytidine (ac4C) is an RNA modification that is catalyzed by the enzyme NAT10. Constitutively found in tRNA and rRNA, ac4C displays a dynamic presence in mRNA that is shaped by developmental and induced shifts in NAT10 levels. However, deciphering ac4C functions in mRNA has been hampered by its context-dependent influences in translation and the complexity of isolating effects on specific mRNAs from other NAT10 activities. Recent advances have begun to overcome these obstacles by leveraging natural variations in mRNA acetylation in cancer, developmental transitions, and immune responses. Here, we synthesize the current literature with a focus on nuances that may fuel the perception of cellular discrepancies toward the development of a cohesive model of ac4C function in mRNA.

Conquering new grounds: plant organellar C-to-U RNA editing factors can be functional in the plant cytosol.

Plant mitochondrial and chloroplast transcripts are subject to numerous events of specific cytidine-to-uridine (C-to-U) RNA editing to correct genetic information. Key protein factors for this process are specific RNA-binding pentatricopeptide repeat (PPR) proteins, which are encoded in the nucleus and post-translationally imported into the two endosymbiotic organelles. Despite hundreds of C-to-U editing sites in the plant organelles, no comparable editing has been found for nucleo-cytosolic mRNAs raising the question why plant RNA editing is restricted to chloroplasts and mitochondria. Here, we addressed this issue in the model moss Physcomitrium patens, where all PPR-type RNA editing factors comprise specific RNA-binding and cytidine deamination functionalities in single proteins. To explore whether organelle-type RNA editing can principally also take place in the plant cytosol, we expressed PPR56, PPR65 and PPR78, three editing factors recently shown to also function in a bacterial setup, together with cytosolic co-transcribed native targets in Physcomitrium. While we obtained unsatisfying results upon their constitutive expression, we found strong cytosolic RNA editing under hormone-inducible expression. Moreover, RNA-Seq analyses revealed varying numbers of up to more than 900 off-targets in other cytosolic transcripts. We conclude that PPR-mediated C-to-U RNA editing is not per se incompatible with the plant cytosol but that its limited target specificity has restricted its occurrence to the much less complex transcriptomes of mitochondria and chloroplast in the course of evolution.

TransAC4C-a novel interpretable architecture for multi-species identification of N4-acetylcytidine sites in RNA with single-base resolution.

N4-acetylcytidine (ac4C) is a modification found in ribonucleic acid (RNA) related to diseases. Expensive and labor-intensive methods hindered the exploration of ac4C mechanisms and the development of specific anti-ac4C drugs. Therefore, an advanced prediction model for ac4C in RNA is urgently needed. Despite the construction of various prediction models, several limitations exist: (1) insufficient resolution at base level for ac4C sites; (2) lack of information on species other than Homo sapiens; (3) lack of information on RNA other than mRNA; and (4) lack of interpretation for each prediction. In light of these limitations, we have reconstructed the previous benchmark dataset and introduced a new dataset including balanced RNA sequences from multiple species and RNA types, while also providing base-level resolution for ac4C sites. Additionally, we have proposed a novel transformer-based architecture and pipeline for predicting ac4C sites, allowing for highly accurate predictions, visually interpretable results and no restrictions on the length of input RNA sequences. Statistically, our work has improved the accuracy of predicting specific ac4C sites in multiple species from less than 40% to around 85%, achieving a high AUC > 0.9. These results significantly surpass the performance of all existing models.

Enhanced ac4C detection in RNA via chemical reduction and cDNA synthesis with modified dNTPs.

The functional analysis of epitranscriptomic modifications in RNA is constrained by a lack of methods that accurately capture their locations and levels. We previously demonstrated that the RNA modification N4-acetylcytidine (ac4C) can be mapped at base resolution through sodium borohydride reduction to tetrahydroacetylcytidine (tetrahydro-ac4C), followed by cDNA synthesis to misincorporate adenosine opposite reduced ac4C sites, culminating in C:T mismatches at acetylated cytidines (RedaC:T). However, this process is relatively inefficient, resulting in <20% C:T mismatches at a fully modified ac4C site in 18S rRNA. Considering that ac4C locations in other substrates including mRNA are unlikely to reach full penetrance, this method is not ideal for comprehensive mapping. Here, we introduce "RetraC:T" (reduction to tetrahydro-ac4C and reverse transcription with amino-dATP to induce C:T mismatches) as a method with enhanced ability to detect ac4C in cellular RNA. In brief, RNA is reduced through NaBH4 or the closely related reagent sodium cyanoborohydride (NaCNBH3) followed by cDNA synthesis in the presence of a modified DNA nucleotide, 2-amino-dATP, that preferentially binds to tetrahydro-ac4C. Incorporation of the modified dNTP substantially improved C:T mismatch rates, reaching stoichiometric detection of ac4C in 18S rRNA. Importantly, 2-amino-dATP did not result in truncated cDNA products nor increase mismatches at other locations. Thus, modified dNTPs are introduced as a new addition to the toolbox for detecting ac4C at base resolution.

N4-acetylcytidine modifies primary microRNAs for processing in cancer cells.

N4 acetylcytidine (ac4C) modification mainly occurs on tRNA, rRNA, and mRNA, playing an important role in the expression of genetic information. However, it is still unclear whether microRNAs have undergone ac4C modification and their potential physiological and pathological functions. In this study, we identified that NAT10/THUMPD1 acetylates primary microRNAs (pri-miRNAs) with ac4C modification. Knockdown of NAT10 suppresses and augments the expression levels of mature miRNAs and pri-miRNAs, respectively. Molecular mechanism studies found that pri-miRNA ac4C promotes the processing of pri-miRNA into precursor miRNA (pre-miRNA) by enhancing the interaction of pri-miRNA and DGCR8, thereby increasing the biogenesis of mature miRNA. Knockdown of NAT10 attenuates the oncogenic characters of lung cancer cells by regulating miRNA production in cancers. Moreover, NAT10 is highly expressed in various clinical cancers and negatively correlated with poor prognosis. Thus, our results reveal that NAT10 plays a crucial role in cancer initiation and progression by modulating pri-miRNA ac4C to affect miRNA production, which would provide an attractive therapeutic strategy for cancers.

Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells.

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddAtox, that can act upon double-stranded DNA. Here, we target DddAtox fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddAtox that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.

N4-acetylcytidine (ac4C) promotes mRNA localization to stress granules.

Stress granules are an integral part of the stress response that are formed from non-translating mRNAs aggregated with proteins. While much is known about stress granules, the factors that drive their mRNA localization are incompletely described. Modification of mRNA can alter the properties of the nucleobases and affect processes such as translation, splicing and localization of individual transcripts. Here, we show that the RNA modification N4-acetylcytidine (ac4C) on mRNA associates with transcripts enriched in stress granules and that stress granule localized transcripts with ac4C are specifically translationally regulated. We also show that ac4C on mRNA can mediate localization of the protein NOP58 to stress granules. Our results suggest that acetylation of mRNA regulates localization of both stress-sensitive transcripts and RNA-binding proteins to stress granules and adds to our understanding of the molecular mechanisms responsible for stress granule formation.

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria.

A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C→T (or G→A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A→G (or T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells.

Recent advances in the potential role of RNA N4-acetylcytidine in cancer progression.

N4-acetylcytidine (ac4C) is a highly conserved chemical modification widely found in eukaryotic and prokaryotic RNA, such as tRNA, rRNA, and mRNA. This modification is significantly associated with various human diseases, especially cancer, and its formation depends on the catalytic activity of N-acetyltransferase 10 (NAT10), the only known protein that produces ac4C. This review discusses the detection techniques and regulatory mechanisms of ac4C and summarizes ac4C correlation with tumor occurrence, development, prognosis, and drug therapy. It also comments on a new biomarker for early tumor diagnosis and prognosis prediction and a new target for tumor therapy. Video Abstract.

Cytidine Acetylation Across the Tree of Life.

Acetylation plays a critical role in regulating eukaryotic transcription via the modification of histones. Beyond this well-documented function, a less explored biological frontier is the potential for acetylation to modify and regulate the function of RNA molecules themselves. N4-Acetylcytdine (ac4C) is a minor RNA nucleobase conserved across all three domains of life (archaea, bacteria, and eukarya), a conservation that suggests a fundamental role in biological processes. Unlike many RNA modifications that are controlled by large enzyme families, almost all organisms catalyze ac4C using a homologue of human Nat10, an essential disease-associated acetyltransferase enzyme.A critical step in defining the fundamental functions of RNA modifications has been the development of methods for their sensitive and specific detection. This Account describes recent progress enabling the use of chemical sequencing reactions to map and quantify ac4C with single-nucleotide resolution in RNA. To orient readers, we first provide historical background of the discovery of ac4C and the enzymes that catalyze its formation. Next, we describe mechanistic experiments that led to the development of first- and second-generation sequencing reactions able to determine ac4C's position in a polynucleotide by exploiting the nucleobase's selective susceptibility to reduction by hydride donors. A notable feature of this chemistry, which may serve as a prototype for nucleotide resolution RNA modification sequencing reactions more broadly, is its ability to drive a penetrant and detectable gain of signal specifically at ac4C sites. Emphasizing practical applications, we present how this optimized chemistry can be integrated into experimental workflows capable of sensitive, transcriptome-wide analysis. Such readouts can be applied to quantitatively define the ac4C landscape across the tree of life. For example, in human cell lines and yeast, this method has uncovered that ac4C is highly selective, predominantly occupying dominant sites within rRNA (rRNA) and tRNA (tRNA). By contrast, when we extend these analyses to thermophilic archaea they identify the potential for much more prevalent patterns of cytidine acetylation, leading to the discovery of a role for this modification in adaptation to environmental stress. Nucleotide resolution analyses of ac4C have also allowed for the determination of structure-activity relationships required for short nucleolar RNA (snoRNA)-catalyzed ac4C deposition and the discovery of organisms with unexpectedly divergent tRNA and rRNA acetylation signatures. Finally, we share how these studies have shaped our approach to evaluating novel ac4C sites reported in the literature and highlight unanswered questions and new directions that set the stage for future research in the field.

Conservation of the moss RNA editing factor PPR78 despite the loss of its known cytidine-to-uridine editing sites is explained by a hidden extra target.

Cytidine (C)-to-uridine (U) RNA editing in plant organelles relies on specific RNA-binding pentatricopeptide repeat (PPR) proteins. In the moss Physcomitrium patens, all such RNA editing factors feature a C-terminal DYW domain that acts as the cytidine deaminase for C-to-U conversion. PPR78 of Physcomitrium targets 2 mitochondrial editing sites, cox1eU755SL and rps14eU137SL. Remarkably, the latter is edited to highly variable degrees in different mosses. Here, we aimed to unravel the coevolution of PPR78 and its 2 target sites in mosses. Heterologous complementation in a Physcomitrium knockout line revealed that the variable editing of rps14eU137SL depends on the PPR arrays of different PPR78 orthologues but not their C-terminal domains. Intriguingly, PPR78 has remained conserved despite the simultaneous loss of editing at both known targets among Hypnales (feather mosses), suggesting it serves an additional function. Using a recently established RNA editing assay in Escherichia coli, we confirmed site-specific RNA editing by PPR78 in the bacterium and identified 4 additional off-targets in the bacterial transcriptome. Based on conservation profiles, we predicted ccmFNeU1465RC as a candidate editing target of PPR78 in moss mitochondrial transcriptomes. We confirmed editing at this site in several mosses and verified that PPR78 targets ccmFNeU1465RC in the bacterial editing system, explaining the conservation and functional adaptation of PPR78 during moss evolution.

Stacking-ac4C: an ensemble model using mixed features for identifying n4-acetylcytidine in mRNA.

N4-acetylcytidine (ac4C) is a modification of cytidine at the nitrogen-4 position, playing a significant role in the translation process of mRNA. However, the precise mechanism and details of how ac4C modifies translated mRNA remain unclear. Since identifying ac4C sites using conventional experimental methods is both labor-intensive and time-consuming, there is an urgent need for a method that can promptly recognize ac4C sites. In this paper, we propose a comprehensive ensemble learning model, the Stacking-based heterogeneous integrated ac4C model, engineered explicitly to identify ac4C sites. This innovative model integrates three distinct feature extraction methodologies: Kmer, electron-ion interaction pseudo-potential values (PseEIIP), and pseudo-K-tuple nucleotide composition (PseKNC). The model also incorporates the robust Cluster Centroids algorithm to enhance its performance in dealing with imbalanced data and alleviate underfitting issues. Our independent testing experiments indicate that our proposed model improves the Mcc by 15.61% and the ROC by 5.97% compared to existing models. To test our model's adaptability, we also utilized a balanced dataset assembled by the authors of iRNA-ac4C. Our model showed an increase in Sn of 4.1%, an increase in Acc of nearly 1%, and ROC improvement of 0.35% on this balanced dataset. The code for our model is freely accessible at https://github.com/louliliang/ST-ac4C.git, allowing users to quickly build their model without dealing with complicated mathematical equations.

LSA-ac4C: A hybrid neural network incorporating double-layer LSTM and self-attention mechanism for the prediction of N4-acetylcytidine sites in human mRNA.

N4-acetylcytidine (ac4C) is a vital constituent of the epitranscriptome and plays a crucial role in the regulation of mRNA expression. Numerous studies have established correlations between ac4C and the incidence, progression and prognosis of various cancers. Therefore, accurately predicting ac4C sites is an important step towards comprehending the biological functions of this modification and devising effective therapeutic interventions. Wet experiments are primary methods for studying ac4C, but computational methods have emerged as a promising supplement due to their cost-effectiveness and shorter research cycles. However, current models still have inherent limitations in terms of predictive performance and generalization ability. Here, we utilized automated machine learning technology to establish a reliable baseline and constructed a deep hybrid neural network, LSA-ac4C, which combines double-layer Long Short-Term Memory (LSTM) and self-attention mechanism for accurate ac4C sites prediction. Benchmarking comparisons demonstrate that LSA-ac4C exhibits superior performance compared to the current state-of-the-art method, with ACC, MCC and AUROC improving by 2.89 %, 5.96 % and 1.53 %, respectively, on an independent test set. Overall, LSA-ac4C serves as a powerful tool for predicting ac4C sites in human mRNA, thus benefiting research on RNA modification. For the convenience of the research community, a web server has been established at http://tubic.org/ac4C.

N4-acetylation of cytidine in mRNA plays essential roles in plants.

The biological function of RNA can be modulated by base modifications. Here, we unveiled the occurrence of N4-acetylation of cytidine in plant RNA, including mRNA, by employing LC-MS/MS and acRIP-seq. We identified 325 acetylated transcripts from the leaves of 4-week-old Arabidopsis (Arabidopsis thaliana) plants and determined that 2 partially redundant N-ACETYLTRANSFERASEs FOR CYTIDINE IN RNA (ACYR1 and ACYR2), which are homologous to mammalian NAT10, are required for acetylating RNA in vivo. A double-null mutant was embryo lethal, while eliminating 3 of the 4 ACYR alleles led to defects in leaf development. These phenotypes could be traced back to the reduced acetylation and concomitant destabilization of the transcript of TOUGH, which is required for miRNA processing. These findings indicate that N4-acetylation of cytidine is a modulator of RNA function with a critical role in plant development and likely many other processes.

PARP-dependent and NAT10-independent acetylation of N4-cytidine in RNA appears in UV-damaged chromatin.

RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs.

Emerging role of RNA acetylation modification ac4C in diseases: Current advances and future challenges.

The oldest known highly conserved modification of RNA, N4-acetylcytidine, is widely distributed from archaea to eukaryotes and acts as a posttranscriptional chemical modification of RNA, contributing to the correct reading of specific nucleotide sequences during translation, stabilising mRNA and improving transcription efficiency. Yeast Kre33 and human NAT10, the only known authors of ac4C, modify tRNA with the help of the Tan1/THUMPD1 adapter to stabilise its structure. Currently, the mRNA for N4-acetylcytidine (ac4C), catalysed by NAT10 (N-acetyltransferase 10), has been implicated in a variety of human diseases, particularly cancer. This article reviews advances in the study of ac4C modification of RNA and the ac4C-related gene NAT10 in normal physiological cell development, cancer, premature disease and viral infection and discusses its therapeutic promise and future research challenges.