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.

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.

A new technique for the analysis of metabolic pathways of cytidine analogues and cytidine deaminase activities in cells.

Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.

NAT10-dependent N4-acetylcytidine modification mediates PAN RNA stability, KSHV reactivation, and IFI16-related inflammasome activation.

N-acetyltransferase 10 (NAT10) is an N4-acetylcytidine (ac4C) writer that catalyzes RNA acetylation at cytidine N4 position on tRNAs, rRNAs and mRNAs. Recently, NAT10 and the associated ac4C have been reported to increase the stability of HIV-1 transcripts. Here, we show that NAT10 catalyzes ac4C addition to the polyadenylated nuclear RNA (PAN), a long non-coding RNA encoded by the oncogenic DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV), triggering viral lytic reactivation from latency. Mutagenesis of ac4C sites in PAN RNA in the context of KSHV infection abolishes PAN ac4C modifications, downregulates the expression of viral lytic genes and reduces virion production. NAT10 knockdown or mutagenesis erases ac4C modifications of PAN RNA and increases its instability, and prevents KSHV reactivation. Furthermore, PAN ac4C modification promotes NAT10 recruitment of IFN-γ-inducible protein-16 (IFI16) mRNA, resulting in its ac4C acetylation, mRNA stability and translation, and eventual inflammasome activation. These results reveal a novel mechanism of viral and host ac4C modifications and the associated complexes as a critical switch of KSHV replication and antiviral immunity.

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.

Cytidine Alleviates Dyslipidemia and Modulates the Gut Microbiota Composition in ob/ob Mice.

Cytidine and uridine are endogenous metabolites in the pyrimidine metabolism pathway, and cytidine is a substrate that can be metabolized into uridine via cytidine deaminase. Uridine has been widely reported to be effective in regulating lipid metabolism. However, whether cytidine could ameliorate lipid metabolism disorder has not yet been investigated. In this research, ob/ob mice were used, and the effect of cytidine (0.4 mg/mL in drinking water for five weeks) on lipid metabolism disorder was evaluated in terms of an oral glucose tolerance test, serum lipid levels, liver histopathological analysis and gut microbiome analysis. Uridine was used as a positive control. Our findings reveal that cytidine could alleviate certain aspects of dyslipidemia and improve hepatic steatosis via modulating the gut microbiota composition in ob/ob mice, especially increasing the abundance of short-chain fatty acids-producing microbiota. These results suggest that cytidine supplementation could be a potential therapeutic approach for dyslipidemia.

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.

Cell-free regeneration of ATP based on polyphosphate kinase 2 facilitates cytidine 5'-monophosphate production.

Cytidine 5'-monophosphate (5'-CMP), a key intermediate for the production of nucleotide derivatives, has been extensively used in food, agriculture, and medicine industries. Compared to RNA degradation and chemical synthesis, the biosynthesis of 5'-CMP has attracted wide attention due to its relatively low cost and eco-friendliness. In this study, we developed a cell-free regeneration of ATP based on polyphosphate kinase 2 (PPK2) to manufacture 5'-CMP from cytidine (CR). McPPK2 from Meiothermus cerbereus exhibited high specific activity (128.5 U/mg) and was used to accomplish ATP regeneration. McPPK2 and LhUCK (a uridine-cytidine kinase from Lactobacillus helveticus) were combined to convert CR to 5'-CMP. Further, the degradation of CR was inhibited by knocking out cdd from the Escherichia coli genome to enhance 5'-CMP production. Finally, the cell-free system based on ATP regeneration maximized the titer of 5'-CMP up to 143.5 mM. The wider applicability of this cell-free system was demonstrated in the synthesis of deoxycytidine 5'-monophosphate (5'-dCMP) from deoxycytidine (dCR) by incorporating McPPK2 and BsdCK (a deoxycytidine kinase from Bacillus subtilis). This study suggests that the cell-free regeneration of ATP based on PPK2 has the advantage of great flexibility for producing 5'-(d)CMP and other (deoxy)nucleotides.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Antiretroviral APOBEC3 cytidine deaminases alter HIV-1 provirus integration site profiles.

APOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. Low levels of deamination are believed to contribute to the genetic evolution of HIV-1, while intense catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total HIV-1 restriction. So far, little is known about how A3 cytosine deaminases might impact HIV-1 proviral DNA integration sites in human chromosomal DNA. Using a deep sequencing approach, we analyze the influence of catalytic active and inactive APOBEC3F and APOBEC3G on HIV-1 integration site selections. Here we show that DNA editing is detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 mutants decrease insertions into gene coding sequences and increase integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicates A3 as a host factor influencing HIV-1 integration site selection and also promotes what appears to be a more latent expression profile.

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.

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.

Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine.

Post-transcriptional RNA editing modulates gene expression in a condition-dependent fashion. We recently discovered C-to-Ψ editing in Vibrio cholerae tRNA. Here, we characterize the biogenesis, regulation, and functions of this previously undescribed RNA editing process. We show that an enzyme, TrcP, mediates the editing of C-to-U followed by the conversion of U to Ψ, consecutively. AlphaFold-2 predicts that TrcP consists of two globular domains (cytidine deaminase and pseudouridylase) and a long helical domain. The latter domain tethers tRNA substrates during both the C-to-U editing and pseudouridylation, likely enabling a substrate channeling mechanism for efficient catalysis all the way to the terminal product. C-to-Ψ editing both requires and suppresses other modifications, creating an interdependent network of modifications in the tRNA anticodon loop that facilitates coupling of tRNA modification states to iron availability. Our findings provide mechanistic insights into an RNA editing process that likely promotes environmental adaptation.

The Combined Escherichia coli Nissle 1917 and Tryptophan Treatment Modulates Immune and Metabolome Responses to Human Rotavirus Infection in a Human Infant Fecal Microbiota-Transplanted Malnourished Gnotobiotic Pig Model.

Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.