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.

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.

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.

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.

ConspectusAcetylation 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.

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.

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.

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.

The Cytidine N-Acetyltransferase NAT10 Participates in Peripheral Nerve Injury-Induced Neuropathic Pain by Stabilizing SYT9 Expression in Primary Sensory Neurons.

RNA N4-acetylcytidine (ac4C) modification is increasingly recognized as an important layer of gene regulation; however, the involvement of ac4C in pain regulation has not been studied. Here, we report that N-acetyltransferase 10 protein (NAT10; the only known ac4C "writer") contributes to the induction and development of neuropathic pain in an ac4C-dependent manner. Peripheral nerve injury increases the levels of NAT10 expression and overall ac4C in injured dorsal root ganglia (DRGs). This upregulation is triggered by the activation of upstream transcription factor 1 (USF1), a transcription factor that binds to the Nat10 promoter. Knock-down or genetic deletion of NAT10 in the DRG abolishes the gain of ac4C sites in Syt9 mRNA and the augmentation of SYT9 protein, resulting in a marked antinociceptive effect in nerve-injured male mice. Conversely, mimicking NAT10 upregulation in the absence of injury evokes the elevation of Syt9 ac4C and SYT9 protein and induces the genesis of neuropathic-pain-like behaviors. These findings demonstrate that USF1-governed NAT10 regulates neuropathic pain by targeting Syt9 ac4C in peripheral nociceptive sensory neurons. Our findings establish NAT10 as a critical endogenous initiator of nociceptive behavior and a promising new target for treating neuropathic pain.SIGNIFICANCE STATEMENT The cytidine N4-acetylcytidine (ac4C), a new epigenetic RNA modification, is crucial for the translation and stability of mRNA, but its role for chronic pain remains unclear. Here, we demonstrate that N-acetyltransferase 10 (NAT10) acts as ac4C N-acetyltransferase and plays an important role in the development and maintenance of neuropathic pain. NAT10 was upregulated via the activation of the transcription factor upstream transcription factor 1 (USF1) in the injured dorsal root ganglion (DRG) after peripheral nerve injury. Since pharmacological or genetic deleting NAT10 in the DRG attenuated the nerve injury-induced nociceptive hypersensitivities partially through suppressing Syt9 mRNA ac4C and stabilizing SYT9 protein level, NAT10 may serve as an effective and novel therapeutic target for neuropathic pain.

Profiling Genome-Wide Specificity of dCpf1 Cytidine Base Editors Using Digenome-Seq.

Digenome-seq is a powerful approach for determining the genome-wide specificity of programmable nuclease including CRISPR-Cas9 and CRISPR-Cpf1 (also known as Cas12a) and programmable deaminase including cytosine base editors (CBEs) and adenine base editors (ABEs). To define the genome-wide specificity of dLbCpf1-BE (also known as dLbCas12a-BE), genomic DNA is first incubated with dLbCpf1-BE, which induces C-to-U conversion at on-target and off-target sites, and then treated with a mixture of E. coli uracil DNA glycosylase (UDG) and Endonuclease VIII, which creates single-strand breaks (SSBs) by removing uracil in vitro. Digested genomic DNA is subjected to WGS, and then sequencing reads are aligned to the reference genome, resulting in straight alignments at on-target and off-target sites. The in vitro cleavage sites related to the straight alignments can be identified using the Digenome-seq computer tool.

iRNA-ac4C: A novel computational method for effectively detecting N4-acetylcytidine sites in human mRNA.

RNA N4-acetylcytidine (ac4C) is the acetylation of cytidine at the nitrogen-4 position, which is a highly conserved RNA modification and involves a variety of biological processes. Hence, accurate identification of genome-wide ac4C sites is vital for understanding regulation mechanism of gene expression. In this work, a novel predictor, named iRNA-ac4C, was established to identify ac4C sites in human mRNA based on three feature extraction methods, including nucleotide composition, nucleotide chemical property, and accumulated nucleotide frequency. Subsequently, minimum-Redundancy-Maximum-Relevance combined with incremental feature selection strategies was utilized to select the optimal feature subset. According to the optimal feature subset, the best ac4C classification model was trained by gradient boosting decision tree with 10-fold cross-validation. The results of independent testing set indicated that our proposed method could produce encouraging generalization capabilities. For the convenience of other researchers, we established a user-friendly web server which is freely available at http://lin-group.cn/server/iRNA-ac4C/. We hope that the tool could provide guide for wet-experimental scholars.

Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

Identification of RBM46 as a novel APOBEC1 cofactor for C-to-U RNA-editing activity.

Cytidine (C) to Uridine (U) RNA editing is a post-transcription modification that is involved in diverse biological processes. APOBEC1 (A1) catalyzes the conversion of C-to-U in RNA, which is important in regulating cholesterol metabolism through its editing activity on ApoB mRNA. However, A1 requires a cofactor to form an "editosome" for RNA editing activity. A1CF and RBM47, both RNA-binding proteins, have been identified as cofactors that pair with A1 to form editosomes and edit ApoB mRNA and other cellular RNAs. SYNCRIP is another RNA-binding protein that has been reported as a potential regulator of A1, although it is not directly involved in A1 RNA editing activity. Here, we describe the identification and characterization of a novel cofactor, RBM46 (RNA-Binding-Motif-protein-46), that can facilitate A1 to perform C-to-U editing on ApoB mRNA. Additionally, using the low-error circular RNA sequencing technique, we identified novel cellular RNA targets for the A1/RBM46 editosome. Our findings provide further insight into the complex regulatory network of RNA editing and the potential new function of A1 with its cofactors.

Revealing the Potential Markers of N(4)-Acetylcytidine through acRIP-seq in Triple-Negative Breast Cancer.

Understanding the causes of tumorigenesis and progression in triple-receptor negative breast cancer (TNBC) can help the design of novel and personalized therapies and prognostic assessments. Abnormal RNA modification is a recently discovered process in TNBC development. TNBC samples from The Cancer Genome Atlas database were categorized according to the expression level of NAT10, which drives acetylation of cytidine in RNA to N(4)-acetylcytidine (ac4C) and affects mRNA stability. A total of 703 differentially expressed long non-coding RNAs (lncRNAs) were found between high- and low-expressed NAT10 groups in TNBC. Twenty of these lncRNAs were significantly associated with prognosis. Two breast cancer tissues and their paired normal tissues were sequenced at the whole genome level using acetylated RNA immunoprecipitation sequencing (acRIP-seq) technology to identify acetylation features in TNBC, and 180 genes were significantly differentially ac4c acetylated in patients. We also analyzed the genome-wide lncRNA expression profile and constructed a co-expression network, containing 116 ac4C genes and 1080 lncRNAs. Three of these lncRNAs were prognostic risk lncRNAs affected by NAT10 and contained in the network. The corresponding reciprocal pairs were "LINC01614-COL3A1", "OIP5-AS1-USP8", and "RP5-908M14.9-TRIR". These results indicate that RNA ac4c acetylation involves lncRNAs and affects the tumor process and prognosis of TNBC. This will aid the prediction of drug targets and drug sensitivity.

Computer-Based Design of a Cell Factory for High-Yield Cytidine Production.

Pyrimidine ribonucleotide de novo biosynthesis pathway (PRdnBP) is an important pathway to produce pyrimidine nucleosides. We attempted to systematically investigate PRdnBP in Escherichia coli with genome-scale metabolic models and utilized the models to guide strain design. The balance of central carbon metabolism and PRdnBP affected the production of cytidine from glucose. Using Bayesian metabolic flux analysis, the effect of modified PRdnBP on the metabolic network was analyzed. The acetate overflow became coupled with PRdnBP flux, while they were originally independent under oxygen-sufficient conditions. The coupling between cytidine production and acetate secretion in the modified strain was weakened by arcA deletion, which resulted in further improving the efficient accumulation of cytidine. In total, 1.28 g/L of cytidine with a yield of 0.26 g/g glucose was produced. The yield of cytidine produced by E. coli is higher than previous reports. Our strategy provides an effective attempt to find metabolic bottlenecks in genetically engineered bacteria by using flux coupling analysis.