RNA modifications as a means of self-recognition and immune protection

Ribonucleotides modifications to mRNA play important roles biological regulations. Over 170 types of RNA modifications have been experimentally validated. Their detection traditionally relies on specific antibody-based enrichment and analytical chemistry tools; these approaches are labor intensive and can detect only one or a few modifications at a time. This is insufficient to truly assess complete transcriptomes for sequence-specific identification and quantitation of epigenetic signals. Recently, we were the first to use third-generation Oxford Nanopore Technology (ONT) sequencing to directly sequence cellular RNA in native from, at a transcriptomic level. We determined that the method can uncover RNA modifications of any type. Based on the principle that such modifications are absent on cDNA or synthetical unmodified RNA, we conducted a study that compared sequence features of native modified RNA with unmodified RNA of the same sequence. We developed a bioinformatics tool, ELIGOS (Epitranscriptional Landscape Inferring from Glitches of ONT Signals), that successfully identified modified RNA bases from the native RNA sequences. ELIGOS accurately predicts known classes of RNA methylation sites (AUC > 0.93) in rRNAs from E. coli, yeast, and human cells, by using either unmodified in vitro transcribed RNA or our developed background-error model, which mimics the systematic error in native RNA sequences. The validity of the approach was illustrated in transcriptomes of yeast, mouse, and human cells. We further apply ELIGOS in detection of DNA adducts and for distinguishing individual alkylated DNA adducts. We analyzed a library of 16 plasmids containing site-specifically inserted O6- or N2-alkyl-deoxyguanosine lesions differing in sizes, functional group, regiochemistries, and abasic site. Based on the native DNA sequences, ELIGOS can accurately identified the location of individual DNA adducts. Moreover, individual DNA adducts were clearly distinguished from each other at the signal level. ELIGOS software is publicly available and can be used to detect possible RNA and DNA modification sites at genome-scale from native RNA/DNA sequences.

RNA modifications in eukaryotic cells have emerged as an exciting but under-explored area in recent years and are considered to be associated with many human diseases. While several studies have been published relating to m6A in osteoarthritis (OA), we only have limited knowledge of other kinds of RNA modifications. Our study investigated eight RNA modifiers' specific roles in OA including A-to-I, APA, m5C, m6A, m7G, mcm5s2U, Nm and Ψ together with their relationship with immune infiltration. RNA modification patterns in OA samples were identified based on eight-type RNA modifiers and their correlation with the degree of immune infiltration was also methodically investigated. Receiver operating characteristic curves (ROC) and qRT-PCR was performed to confirm the abnormal expression of hub genes. The RNA modification score (Rmscore) was generated by the applications of principal component analysis (PCA) algorithm in order to quantify RNA modification modes in individual OA patients. We identified 21 differentially-expressed RNA modification related genes between OA and healthy samples. For example, CFI, CBLL1 and ALKBH8 were expressed at high levels in OA (P<0.001), while RPUSD4, PUS1, NUDT21, FBL and WDR4 were expressed at low levels (P<0.001). Two candidate RNA modification regulators (WDR4 and CFI) were screened out utilizing a random forest machine learning model. We then identified two distinctive RNA modification modes in OA which were found to display distinctive biological features. High Rmscore, characterized by increased immune cell infiltration, indicated an inflamed phenotype. Our study was the first to systematically reveal the crosstalk and dysregulations eight-type of RNA modifications in OA. Assessing individuals' RNA modification patterns will be conductive to enhance our understanding of the properties of immune infiltration, provide novel diagnostic and prognostic biomarkers, and guide more effective immunotherapy strategies in the future.

ABSTRACTAmong myriads of distinct chemical modifications in RNAs, dynamic N6-methyladenosine (m6A) is one of the most prevalent modifications in eukaryotic mRNAs and non-coding RNAs. Similar to the critical role of chemical modifications in regulation of DNA and protein activities, RNA m6A modification is also observed to be involved in the regulation of diverse functions of RNAs including meiosis, fertility, development, cell reprogramming and circadian period. The RNA m6A modification is recognized by YTH domain containing family proteins comprising of YTHDC1-2 and YTHDF1-3. Here we focus on the nuclear m6A reader YTHDC1 and its regulatory role in alternative splicing and other RNA metabolic processes.

Molecular Regulation, Evolutionary, and Functional Adaptations Associated with C to U Editing of Mammalian ApolipoproteinB mRNA

Fibroblasts play an important role in the pathogenic mechanisms of several socially significant diseases, including pulmonary and cardiovascular fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease. The alterations of the epitranscriptome, including more than 170 distinct posttranscriptional RNA modifications or editing events, justified their investigation as an important modulator of fibrosis. Recent development of high-throughput methods allows the identification of RNA modification sites and their mechanistic aspect in the fibrosis development. The most common RNA modification is methylation of N6-adenosine deposited by the m6A methyltransferase complex (METTL3/14/16, WTAP, KIAA1429, and RBM15/15B), erased by demethylases (FTO and ALKBH5), and recognized by binding proteins (e.g., YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, etc.). Adenosine to inosine (A-to-I) RNA editing is another abundant editing event converting adenosine to inosine in double-stranded RNA regions through the action of the adenosine deaminase (ADAR) proteins. Last but not least, 5-methylcytosine (m5C) regulates the stability and translation of mRNAs. All those RNA modifications have been observed in mRNA as well as the noncoding regions of pre-mRNA and noncoding RNAs (ncRNAs) and demonstrated to be involved in fibrosis in different cellular and animal models. This Mini-Review focuses on the latest research on epitranscriptomic marks related to fibroblast biology and fibrosis as well as elucidates the future research directions in this context.

RNA modifications found in most RNAs, particularly in tRNAs and rRNAs, reveal an abundance of chemical alterations of nucleotides. Over 150 distinct RNA modifications are known, emphasizing a remarkable diversity of chemical moieties in RNA molecules. These modifications play pivotal roles in RNA maturation, structural integrity, and the fidelity and efficiency of translation processes. The catalysts responsible for these modifications are RNA-modifying enzymes that use a striking array of chemistries to directly influence the chemical landscape of RNA. This diversity is further underscored by instances where the same modification is introduced by distinct enzymes that use unique catalytic mechanisms and cofactors across different domains of life. This phenomenon of convergent evolution highlights the biological importance of RNA modification and the vast potential within the chemical repertoire for nucleotide alteration. While shared RNA modifications can hint at conserved enzymatic pathways, a major bottleneck is to identify alternative routes within species that possess a modified RNA but are devoid of known RNA-modifying enzymes. To address this challenge, a combination of bioinformatic and experimental strategies proves invaluable in pinpointing new genes responsible for RNA modifications. This integrative approach not only unveils new chemical insights but also serves as a wellspring of inspiration for biocatalytic applications and drug design. In this Account, we present how comparative genomics and genome mining, combined with biomimetic synthetic chemistry, biochemistry, and anaerobic crystallography, can be judiciously implemented to address unprecedented and alternative chemical mechanisms in the world of RNA modification. We illustrate these integrative methodologies through the study of tRNA and rRNA modifications, dihydrouridine, 5-methyluridine, queuosine, 8-methyladenosine, 5-carboxymethylamino-methyluridine, or 5-taurinomethyluridine, each dependent on a diverse array of redox chemistries, often involving organic compounds, organometallic complexes, and metal coenzymes. We explore how vast genome and tRNA databases empower comparative genomic analyses and enable the identification of novel genes that govern RNA modification. Subsequently, we describe how the isolation of a stable reaction intermediate can guide the synthesis of a biomimetic to unveil new enzymatic pathways. We then discuss the usefulness of a biochemical "shunt" strategy to study catalytic mechanisms and to directly visualize reactive intermediates bound within active sites. While we primarily focus on various RNA-modifying enzymes studied in our laboratory, with a particular emphasis on the discovery of a SAM-independent methylation mechanism, the strategies and rationale presented herein are broadly applicable for the identification of new enzymes and the elucidation of their intricate chemistries. This Account offers a comprehensive glimpse into the evolving landscape of RNA modification research and highlights the pivotal role of integrated approaches to identify novel enzymatic pathways.

RNA editing of mitochondrial gene transcripts plays a central role during plant development and evolutionary adaptation. RNA editing has previously been reported to differ between the rice cytoplasmic male sterile (CMS) line and its maintainer line, which has been suggested as a cause for their different performances under environmental stress. To specifically test this hypothesis, a wild abortive (WA) CMS line (Huhan-1A) and its maintainer line (Huhan-1B) were utilized to investigate performances in response to oxidative stress, as well as RNA editing efficiencies on transcripts of six selected mitochondrial genes. Compared to the maintainer line, Huhan-1A represented both lower plant height and total antioxidant capacity, possessed higher total soluble protein and chlorophyll contents, accumulated less H2O2 content on the 3rd day after treatment (DAT), and exhibited higher survival ratio after re-watering. Furthermore, a total of 90 editing sites were detected on transcripts of six mitochondrial genes (atp9, nad2, nad7, nad9, ccmB, and ccmC) in both Huhan-1A and Huhan-1B on the 0, 1st, and 3rd DAT. Forty-eight sites were furthermore determined as stress-responsive sites (SRS). Generally, in response to oxidative stress, SRS in Huhan-1A increased the resulting editing efficiencies, while SRS in Huhan-1B decreased the resulting editing efficiencies. In addition, 33 and 22 sites at ccmB and ccmC were differentially edited between Huhan-1A and Huhan-1B, respectively, on the 0, 1st, and 3rd DAT. Editing efficiencies of ccmB and ccmC were generally lower in Huhan-1A (ccmB, 37.3–47.8%; ccmC, 41.2–52.3%) than those in Huhan-1B (ccmB, 82.6–86.5%; ccmC, 81.0–82.9%). Deficiencies of RNA editing in Huhan-1A at ccmB and ccmC could lead to the loss of transmembrane domains in their protein structures. Consequently, differences in RNA editing at ccmB and ccmC between the WA-CMS line and its maintainer line partially explained their different performances under stress. Moreover, we detected differences in expressions of pentatricopeptide repeat (PPR) genes between both lines, as well as significant correlations with RNA editing. Our study indicated potential associations of RNA editing and PPR genes in rice tolerance to abiotic stresses. However, the underlying molecular mechanisms of stress-adaptation, which are attributed to RNA editing on transcripts of mitochondrial genes, require further investigation.

BackgroundProtein recoding by RNA editing is required for normal health and evolutionary adaptation. However, de novo induction of RNA editing in response to environmental factors is an uncommon phenomenon. While APOBEC3A edits many mRNAs in monocytes and macrophages in response to hypoxia and interferons, the physiological significance of such editing is unclear.ResultsHere, we show that the related cytidine deaminase, APOBEC3G, induces site-specific C-to-U RNA editing in natural killer cells, lymphoma cell lines, and, to a lesser extent, CD8-positive T cells upon cellular crowding and hypoxia. In contrast to expectations from its anti-HIV-1 function, the highest expression of APOBEC3G is shown to be in cytotoxic lymphocytes. RNA-seq analysis of natural killer cells subjected to cellular crowding and hypoxia reveals widespread C-to-U mRNA editing that is enriched for genes involved in mRNA translation and ribosome function. APOBEC3G promotes Warburg-like metabolic remodeling in HuT78 T cells under similar conditions. Hypoxia-induced RNA editing by APOBEC3G can be mimicked by the inhibition of mitochondrial respiration and occurs independently of HIF-1α.ConclusionsAPOBEC3G is an endogenous RNA editing enzyme in primary natural killer cells and lymphoma cell lines. This RNA editing is induced by cellular crowding and mitochondrial respiratory inhibition to promote adaptation to hypoxic stress.

Among over 150 distinct RNA modifications, N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) RNA editing represent 2 of the most studied modifications on mammalian mRNAs. Although both modifications occur on adenosine residues, knowledge on potential functional crosstalk between these 2 modifications is still limited. Here, we show that the m6A modification promotes expression levels of the ADAR1, which encodes an A-to-I RNA editing enzyme, in response to interferon (IFN) stimulation. We reveal that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) mediates up-regulation of ADAR1; YTHDF1 is a reader protein that can preferentially bind m6A-modified transcripts and promote translation. Knockdown of YTHDF1 reduces the overall levels of IFN-induced A-to-I RNA editing, which consequently activates dsRNA-sensing pathway and increases expression of various IFN-stimulated genes. Physiologically, YTHDF1 deficiency inhibits virus replication in cells through regulating IFN responses. The A-to-I RNA editing activity of ADAR1 plays important roles in the YTHDF1-dependent IFN responses. Therefore, we uncover that m6A and YTHDF1 affect innate immune responses through modulating the ADAR1-mediated A-to-I RNA editing.

Gastric cancer (GC) is one of the most common neoplastic malignancies, which permutes a fourth of cancer-related mortality globally. RNA modification plays a significant role in tumorigenesis, the underlying molecular mechanism of how different RNA modifications directly affect the tumor microenvironment (TME) in GC is unclear. Here, we profiled the genetic and transcriptional alterations of RNA modification genes (RMGs) in GC samples from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. Through the unsupervised clustering algorithm, we identified three distinct RNA modification clusters and found that they participate in different biological pathways and starkly correlate with the clinicopathological characteristics, immune cell infiltration, and prognosis of GC patients. Subsequently, univariate Cox regression analysis unveiled 298 of 684 subtype-related differentially expressed genes (DEGs) are tightly interwoven to prognosis. In addition, we conducted the principal component analysis to develop the RM_Score system, which was used to quantify and predict the prognostic value of RNA modification in GC. Our analysis indicated that patients with high RM_Score were characterized by higher tumor mutational burden, mutation frequency, and microsatellite instability which were more susceptible to immunotherapy and had a favorable prognosis. Altogether, our study uncovered RNA modification signatures that may have a potential role in the TME and prediction of clinicopathological characteristics. Identification of these RNA modifications may provide a new understanding of immunotherapy strategies for gastric cancer.

RNA modifications are ubiquitous in host cells and hold crucial roles in regulating diverse biological processes. Although the m6A modification during viral infections has garnered significant attention, the involvement of other modifications in such infections remains underexplored. This study was aimed to map out the comprehensive RNA modification profiles in porcine kidney PK-15 cells infected with pseudorabies virus (PRV) at various time points. Throughout the PRV infection, we identified a total of 31 distinct RNA modifications, among which 25 were detected in significantly higher abundances. Our findings indicated that PRV infection largely suppressed the overall level of RNA modifications, but interestingly, it elevated specific modifications, namely Am, Cm, Gm, and Um. Upon deeper investigation, we discovered that PRV infection notably diminished the levels of enzymes related to m1A modification. Collectively, this study uncovers the landscapes of RNA modification in PRV infection, paving the way for further explorations into RNA modifications during herpesvirus infections.

Pancreatic cancer (PC) is one of the most lethal malignancies and carries a dismal mortality and morbidity. Four types of RNA modification (namely m6A, m1A, APA and A-to-I) could be catalyzed by distinct enzymatic compounds ("writers"), mediating numerous epigenetic events in carcinogenesis and immunomodulation. We aim to investigate the interplay mechanism of these writers in immunogenomic features and molecular biological characteristics in PC. We first accessed the specific expression pattern and transcriptional variation of 26 RNA modification writers in The Cancer Genome Atlas (TCGA) dataset. Unsupervised consensus clustering was performed to divide patients into two RNA modification clusters. Then, based on the differentially expressed genes (DEGs) among two clusters, RNA modification score (WM_Score) model was established to determine RNA modification-based subtypes and was validated in International Cancer Genome Consortium (ICGC) dataset. What's more, we manifested the unique status of WM_Score in transcriptional and post-transcriptional regulation, molecular biological characteristics, targeted therapies and immunogenomic patterns. We documented the tight-knit correlations between transcriptional expression and variation of RNA modification writers. We classified patients into two distinct RNA modification patterns (WM_Score_high and _low), The WM_Score_high subgroup was correlated with worse prognosis, Th2/Th17 cell polarization and oncogenic pathways (e.g. EMT, TGF-β, and mTORC1 signaling pathways), whereas the WM_Score_low subgroup associated with favorable survival rate and Th1 cell trend. WM_Score model also proved robust predictive power in interpreting transcriptional and post-transcriptional events. Additionally, the potential targeted compounds with related pathways for the WM_Score model were further identified. Our research unfolds a novel horizon on the interplay network of four RNA modifications in PC. This WM_Score model demonstrated powerful predictive capacity in epigenetic, immunological and biological landscape, providing a theoretical basis for future clinical judgments of PC.

BackgroundThe four major RNA adenosine modifications, i.e., m6A, m1A, alternative polyadenylation, and adenosine-to-inosine RNA editing, are mediated mostly by the “writer” enzymes and constitute critical mechanisms of epigenetic regulation in immune response and tumorigenesis. However, the cross-talk and potential roles of these “writers” in the tumor microenvironment (TME), drug sensitivity, and immunotherapy remain unknown.MethodsWe systematically characterized mRNA expression and genetic alterations of 26 RNA modification “writers” in colorectal cancer (CRC), and evaluated their expression pattern in 1697 CRC samples from 8 datasets. We used an unsupervised clustering method to assign the samples into two patterns of expression of RNA modification “writers”. Subsequently, we constructed the RNA modification “writer” Score (WM_Score) model based on differentially expressed genes (DEGs) responsible for the RNA modification patterns to quantify the RNA modification-related subtypes of individual tumors. Furthermore, we performed association analysis for WM_Score and characteristics of TME, consensus molecular subtypes (CMSs), clinical features, transcriptional and post-transcriptional regulation, drug response, and the efficacy of immunotherapy.ResultsWe demonstrated that multi-layer alterations of RNA modification “writer” are associated with patient survival and TME cell-infiltrating characteristics. We identified two distinct RNA modification patterns, characterized by a high and a low WM_Score. The WM_Score-high group was associated with worse patient overall survival and with the infiltration of inhibitory immune cells, such as M2 macrophages, EMT activation, and metastasis, while the WM_Score-low group was associated with a survival advantage, apoptosis, and cell cycle signaling pathways. WM_Score correlated highly with the regulation of transcription and post-transcriptional events contributing to the development of CRC. In response to anti-cancer drugs, WM_Score highly negatively correlated (drug sensitive) with drugs which targeted oncogenic related pathways, such as MAPK, EGFR, and mTOR signaling pathways, positively correlated (drug resistance) with drugs which targeted in apoptosis and cell cycle. Importantly, the WM_Score was associated with the therapeutic efficacy of PD-L1 blockade, suggesting that the development of potential drugs targeting these “writers” to aid the clinical benefits of immunotherapy.ConclusionsOur study is the first to provide a comprehensive analysis of four RNA modifications in CRC. We revealed the potential function of these writers in TME, transcriptional and post-transcriptional events, and identified their therapeutic liability in targeted therapy and immunotherapy. This work highlights the cross-talk and potential clinical utility of RNA modification “writers” in cancer therapy.

Background: Bladder cancer (BCa) is the leading reason for death among genitourinary malignancies. RNA modifications in tumors closely link to the immune microenvironment. Our study aimed to propose a promising model associated with the “writer” enzymes of five primary RNA adenosine modifications (including m6A, m6Am, m1A, APA, and A-to-I editing), thus characterizing the clinical outcome, immune landscape and therapeutic efficacy of BCa.Methods: Unsupervised clustering was employed to categorize BCa into different RNA modification patterns based on gene expression profiles of 34 RNA modification “writers”. The RNA modification “writers” score (RMS) signature composed of RNA phenotype-associated differentially expressed genes (DEGs) was established using the least absolute shrinkage and selection operator (LASSO), which was evaluated in meta-GEO (including eight independent GEO datasets) training cohort and the TCGA-BLCA validation cohort. The hub genes in the RMS model were determined via weighted gene co-expression network analysis (WGCNA) and were further validated using human specimen. The potential applicability of the RMS model in predicting the therapeutic responsiveness was assessed through the Genomics of Drug Sensitivity in Cancer database and multiple immunotherapy datasets.Results: Two distinct RNA modification patterns were determined among 1,410 BCa samples from a meta-GEO cohort, showing radically varying clinical outcomes and biological characteristics. The RMS model comprising 14 RNA modification phenotype-associated prognostic DEGs positively correlated with the unsatisfactory outcome of BCa patients in meta-GEO training cohort (HR = 3.00, 95% CI = 2.19–4.12) and TCGA-BLCA validation cohort (HR = 1.53, 95% CI = 1.13–2.09). The infiltration of immunosuppressive cells and the activation of EMT, angiogenesis, IL-6/JAK/STAT3 signaling were markedly enriched in RMS-high group. A nomogram exhibited high prognostic prediction accuracy, with a concordance index of 0.785. The therapeutic effect of chemotherapeutic agents and antibody-drug conjugates was significantly different between RMS-low and -high groups. The combination of the RMS model and conventional characteristics (TMB, TNB and PD-L1) achieved an optimal AUC value of 0.828 in differentiating responders from non-responders to immunotherapy.Conclusion: We conferred the first landscape of five forms of RNA modifications in BCa and emphasized the excellent power of an RNA modifications-related model in evaluating BCa prognosis and immune landscape.

Gene expression is regulated at multiple levels beyond genetic and epigenetic modifications of DNA. Even when DNA is accurately transcribed into RNA, transcript abundance often does not correlate with protein levels, underscoring the importance of posttranscriptional regulation. Chemical modifications of RNA occur in both coding and noncoding RNAs and add an additional layer of gene control. More than 170 distinct RNA modifications have been identified across different RNAs in all domains of life. Although the abundant modifications have been investigated, their roles in cardiac biology and heart failure remain incompletely defined. This review discusses the current knowledge of both well characterized and understudied RNA modifications in the heart. Recent studies highlight context-dependent roles of N6-methyladenosine (m6A), 5-methylcytosine (m5C), N4-acetylcytidine (ac4C), and adenosine-to-inosine (A-to-I) RNA editing in the heart are discussed in this review. RNA modifications constitute a critical regulatory layer that complements transcriptional control and facilitates rapid adaptation to cardiac stress. Dysregulation of m6A, m5C, and A-to-I editing contributes to pathological remodeling and disease progression. However, most RNA modifications remain unexplored in heart failure. Enzymes that write, erase, or read these marks represent promising targets for precision therapeutic strategies.