Comprehensive of N1-Methyladenosine Modifications Patterns and Immunological Characteristics in Ovarian Cancer.

Background: recently, many researches have concentrated on the relevance between N1-methyladenosine (m1A) methylation modifications and tumor progression and prognosis. However, it remains unknown whether m1A modification has an effect in the prognosis of ovarian cancer (OC) and its immune infiltration. Methods: Based on 10 m1A modulators, we comprehensively assessed m1A modification patterns in 474 OC patients and linked them to TME immune infiltration characteristics. m1Ascore computed with principal component analysis algorithm was applied to quantify m1A modification pattern in OC patients. m1A regulators protein and mRNA expression were respectively obtained by HPA website and RT-PCR in clinical OC and normal samples. Results: We finally identified three different m1A modification patterns. The immune infiltration features of these m1A modification patterns correspond to three tumor immune phenotypes, including immune-desert, immune-inflamed and immune-excluded phenotypes. The results demonstrate individual tumor m1A modification patterns can predict patient survival, stage and grade. The m1Ascore was calculated to quantify individual OC patient's m1A modification pattern. A high m1Ascore is usually accompanied by a better survival advantage and a lower mutational load. Research on m1Ascore in the treatment of OC patients showed that patients with high m1Ascore showed marked therapeutic benefits and clinical outcomes in terms of chemotherapy and immunotherapy. Lastly, we obtained four small molecule drugs that may potentially ameliorate prognosis. Conclusion: This research demonstrates that m1A methylation modification makes an essential function in the prognosis of OC and in shaping the immune microenvironment. Comprehensive evaluation of m1A modifications improves our knowledge of immune infiltration profile and provides a more efficient individualized immunotherapy strategy for OC patients.

RNA N6 -methyladenosine modification in the lethal teamwork of cancer stem cells and the tumor immune microenvironment: Current landscape and therapeutic potential.

N6 -methyladenosine (m6 A), the newest and most prevalent layer of internal epigenetic modification in eukaryotic mRNA, has been demonstrated to play a critical role in cancer biology. Increasing evidence has highlighted that the interaction between cancer stem cells (CSCs) and the tumor immune microenvironment (TIME) is the root cause of tumorigenesis, metastasis, therapy resistance, and recurrence. In recent studies, the m6 A modification has been tightly linked to this CSC-TIME interplay, participating in the regulation of CSCs and TIME remolding. Interestingly, the m6 A modification has also been identified as a novel decisive factor in the efficacy of immunotherapies-particularly anti-PD-1/PD-L1 monotherapies-by changing the plasticity of the TIME. Given the functional importance of the m6 A modification in the crosstalk between CSCs and the TIME, targeting m6 A regulators will open new avenues to overcome therapeutic resistance, especially for immune checkpoint-based immunotherapy. In the present review, we summarize the current landscape of m6 A modifications in CSCs and the TIME, and also prospect the underling role of m6 A modifications at the crossroads of CSCs and the TIME for the first time. Additionally, to provide the possibility of modulating m6 A modifications as an emerging therapeutic strategy, we also explore the burgeoning inhibitors and technologies targeting m6 A regulators. Lastly, considering recent advances in m6 A-seq technologies and cancer drug development, we propose the future directions of m6 A modification in clinical applications, which may not only help to improve individualized monitoring and therapy but also provide enhanced and durable responses in patients with insensitive tumors.

Epigenetic regulation in antiviral innate immunity.

Emerging life-threatening viruses have posed great challenges to public health. It is now increasingly clear that epigenetics plays a role in shaping host-virus interactions and there is a great need for a more thorough understanding of these intricate interactions through the epigenetic lens, which may represent potential therapeutic opportunities in the clinic. In this review, we highlight the current understanding of the roles of key epigenetic regulators - chromatin remodeling and histone modification - in modulating chromatin openness during host defense against virus. We also discuss how the RNA modification m6A (N6-methyladenosine) affects fundamental aspects of host-virus interactions. We conclude with future directions for uncovering more detailed functions that epigenetic regulation exerts on both host cells and viruses during infection.

A Posttranscriptional Pathway of CD40 Ligand mRNA Stability Is Required for the Development of an Optimal Humoral Immune Response.

CD40 ligand (CD40L) mRNA stability is dependent on an activation-induced pathway that is mediated by the binding complexes containing the multifunctional RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1) to a 3' untranslated region of the transcript. To understand the relationship between regulated CD40L and the requirement for variegated expression during a T-dependent response, we engineered a mouse lacking the CD40L stability element (CD40LΔ5) and asked how this mutation altered multiple aspects of the humoral immunity. We found that CD40LΔ5 mice expressed CD40L at 60% wildtype levels, and lowered expression corresponded to significantly decreased levels of T-dependent Abs, loss of germinal center (GC) B cells and a disorganized GC structure. Gene expression analysis of B cells from CD40LΔ5 mice revealed that genes associated with cell cycle and DNA replication were significantly downregulated and genes linked to apoptosis upregulated. Importantly, somatic hypermutation was relatively unaffected although the number of cells expressing high-affinity Abs was greatly reduced. Additionally, a significant loss of plasmablasts and early memory B cell precursors as a percentage of total GL7+ B cells was observed, indicating that differentiation cues leading to the development of post-GC subsets was highly dependent on a threshold level of CD40L. Thus, regulated mRNA stability plays an integral role in the optimization of humoral immunity by allowing for a dynamic level of CD40L expression on CD4 T cells that results in the proliferation and differentiation of pre-GC and GC B cells into functional subsets.

Post-transcriptional control of T-cell cytokine production: Implications for cancer therapy.

As part of the adaptive immune system, T cells are vital for the eradication of infected and malignantly transformed cells. To perform their protective function, T cells produce effector molecules that are either directly cytotoxic, such as granzymes, perforin, interferon-γ and tumour necrosis factor α, or attract and stimulate (immune) cells, such as interleukin-2. As these molecules can also induce immunopathology, tight control of their production is required. Indeed, inflammatory cytokine production is regulated on multiple levels. Firstly, locus accessibility and transcription factor availability and activity determine the amount of mRNA produced. Secondly, post-transcriptional mechanisms, influencing mRNA splicing/codon usage, stability, decay, localization and translation rate subsequently determine the amount of protein that is produced. In the immune suppressive environments of tumours, T cells gradually lose the capacity to produce effector molecules, resulting in tumour immune escape. Recently, the role of post-transcriptional regulation in fine-tuning T-cell effector function has become more appreciated. Furthermore, several groups have shown that exhausted or dysfunctional T cells from cancer patients or murine models possess mRNA for inflammatory mediators, but fail to produce effector molecules, hinting that post-transcriptional events also play a role in hampering tumour-infiltrating lymphocyte effector function. Here, the post-transcriptional regulatory events governing T-cell cytokine production are reviewed, with a specific focus on the importance of post-transcriptional regulation in anti-tumour responses. Furthermore, potential approaches to circumvent tumour-mediated dampening of T-cell effector function through the (dis)engagement of post-transcriptional events are explored, such as CRISPR/Cas9-mediated genome editing or chimeric antigen receptors.

N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages.

N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro. HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.

Loss of the Transfer RNA Wobble Uridine-Modifying Enzyme Elp3 Delays T Cell Cycle Entry and Impairs T Follicular Helper Cell Responses through Deregulation of Atf4.

The activation of T cells is accompanied by intensive posttranscriptional remodeling of their proteome. We observed that protein expression of enzymes that modify wobble uridine in specific tRNAs, namely elongator subunit 3 (Elp3) and cytosolic thiouridylase (Ctu)2, increased in the course of T cell activation. To investigate the role of these tRNA epitranscriptomic modifiers in T cell biology, we generated mice deficient for Elp3 in T cells. We show that deletion of Elp3 has discrete effects on T cells. In vitro, Elp3-deficient naive CD4+ T cells polarize normally but are delayed in entering the first cell cycle following activation. In vivo, different models of immunization revealed that Elp3-deficient T cells display reduced expansion, resulting in functional impairment of T follicular helper (TFH) responses, but not of other CD4+ effector T cell responses. Transcriptomic analyses identified a progressive overactivation of the stress-responsive transcription factor Atf4 in Elp3-deficient T cells. Overexpression of Atf4 in wild-type T cells phenocopies the effect of Elp3 loss on T cell cycle entry and TFH cell responses. Reciprocally, partial silencing of Atf4 or deletion of its downstream effector transcription factor Chop rescues TFH responses of Elp3-deficient T cells. Together, our results reveal that specific epitranscriptomic tRNA modifications contribute to T cell cycle entry and promote optimal TFH responses.

Bacterial tRNA 2'-O-methylation is dynamically regulated under stress conditions and modulates innate immune response.

RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2'-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.

The RNA modification N6-methyladenosine as a novel regulator of the immune system.

Protection from harmful pathogens depends on activation of the immune system, which relies on tight regulation of gene expression. Recently, the RNA modification N6-methyladenosine (m6A) has been found to play an essential role in such regulation. Here, we summarize newly discovered functions of m6A in controlling various aspects of immunity, including immune recognition, activation of innate and adaptive immune responses, and cell fate decisions. We then discuss some of the current challenges in the field and describe future directions for uncovering the immunological functions of m6A and its mechanisms of action.

Dynamic Post-Transcriptional Events Governing CD8+ T Cell Homeostasis and Effector Function.

Effective T cell responses against infections and tumors require a swift and ample production of cytokines, chemokines, and cytotoxic molecules. The production of these effector molecules relies on rapid changes of gene expression, determined by cell-intrinsic signals and environmental cues. Here, we review our current understanding of gene-specific regulatory networks that define the magnitude and timing of cytokine production in CD8+ T cells. We discuss the dynamic features of post-transcriptional control during CD8+ T cell homeostasis and activation, and focus on the crosstalk between cell signaling and RNA-binding proteins. Elucidating gene-specific regulatory circuits may help in the future to rectify dysfunctional T cell responses.

Approaches to study CRISPR RNA biogenesis and the key players involved.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide an inheritable and adaptive immune system against phages and foreign genetic elements in many bacteria and archaea. The three stages of CRISPR-Cas immunity comprise adaptation, CRISPR RNA (crRNA) biogenesis and interference. The maturation of the pre-crRNA into mature crRNAs, short guide RNAs that target invading nucleic acids, is crucial for the functionality of CRISPR-Cas defense systems. Mature crRNAs assemble with Cas proteins into the ribonucleoprotein (RNP) effector complex and guide the Cas nucleases to the cognate foreign DNA or RNA target. Experimental approaches to characterize these crRNAs, the specific steps toward their maturation and the involved factors, include RNA-seq analyses, enzyme assays, methods such as cryo-electron microscopy, the crystallization of proteins, or UV-induced protein-RNA crosslinking coupled to mass spectrometry analysis. Complex and multiple interactions exist between CRISPR-cas-encoded specific riboendonucleases such as Cas6, Cas5d and Csf5, endonucleases with dual functions in maturation and interference such as the enzymes of the Cas12 and Cas13 families, and nucleases belonging to the cell's degradosome such as RNase E, PNPase and RNase J, both in the maturation as well as in interference. The results of these studies have yielded a picture of unprecedented diversity of sequences, enzymes and biochemical mechanisms.

Posttranslational Regulation of IL-23 Production Distinguishes the Innate Immune Responses to Live Toxigenic versus Heat-Inactivated Vibrio cholerae.

Vibrio cholerae infection provides long-lasting protective immunity, while oral, inactivated cholera vaccines (OCV) result in more-limited protection. To identify characteristics of the innate immune response that may distinguish natural V. cholerae infection from OCV, we stimulated differentiated, macrophage-like THP-1 cells with live versus heat-inactivated V. cholerae with and without endogenous or exogenous cholera holotoxin (CT). Interleukin 23A gene (IL23A) expression was higher in cells exposed to live V. cholerae than in cells exposed to inactivated organisms (mean change, 38-fold; 95% confidence interval [95% CI], 4.0 to 42; P < 0.01). IL-23 secretion was also higher in cells exposed to live V. cholerae than in cells exposed to inactivated V. cholerae (mean change, 5.6-fold; 95% CI, 4.4 to 11; P < 0.001). This increase in IL-23 secretion was more marked than for other key innate immune cytokines (e.g., IL-1ß and IL-6) and dependent on exposure to the combination of both live V. cholerae and CT. While IL-23 secretion was reduced following stimulation with either heat-inactivated wild-type V. cholerae or a live isogenic ctxAB mutant of V. cholerae, the addition of exogenous CT restored IL-23 secretion in combination with the live isogenic ctxAB mutant V. cholerae, but not when it was paired with stimulation by heat-inactivated V. cholerae The posttranslational regulation of IL-23 under these conditions was dependent on the activity of the cysteine protease cathepsin B. In humans, IL-23 promotes the differentiation of Th17 cells to T follicular helper cells, which maintain and support long-term memory B cell generation after infection. Based on these findings, the stimulation of IL-23 production may be a determinant of protective immunity following V. cholerae infection.IMPORTANCE An episode of cholera provides better protection against reinfection than oral cholera vaccines, and the reasons for this are still under study. To better understand this, we compared the immune responses of human cells exposed to live Vibrio cholerae with those of cells exposed to heat-killed V. cholerae (similar to the contents of oral cholera vaccines). We also compared the effects of active cholera toxin and the inactive cholera toxin B subunit (which is included in some cholera vaccines). One key immune signaling molecule, IL-23, was uniquely produced in response to the combination of live bacteria and active cholera holotoxin. Stimulation with V. cholerae that did not produce the active toxin or was killed did not produce an IL-23 response. The stimulation of IL-23 production by cholera toxin-producing V. cholerae may be important in conferring long-term immunity after cholera.

Re-evaluating Strategies to Define the Immunoregulatory Roles of miRNAs.

miRNAs play an important role in fine-tuning host immune homeostasis and responses through the regulation of mRNA stability and translation. Studies have demonstrated that miRNA-mediated regulation of gene expression has a profound impact on immune cell development, function, and response to invading pathogens. As we continue to examine the mechanisms by which miRNAs maintain the balance between robust protective host immune responses and dysregulated responses that promote immune pathology, careful consideration of the complexity of post-transcriptional immune regulation is needed. Distinct tissue- and stimulus-specific RNA-RNA and RNA-protein interactions can modulate the functions of a given miRNA. Thus, new challenges emerge in the identification of post-transcriptional coregulatory modules and the genetic factors that impact miRNA function.

GAPDH Binding to TNF-α mRNA Contributes to Posttranscriptional Repression in Monocytes: A Novel Mechanism of Communication between Inflammation and Metabolism.

Expression of the inflammatory cytokine TNF is tightly controlled. During endotoxin tolerance, transcription of TNF mRNA is repressed, although not entirely eliminated. Production of TNF cytokine, however, is further controlled by posttranscriptional regulation. In this study, we detail a mechanism of posttranscriptional repression of TNF mRNA by GAPDH binding to the TNF 3' untranslated region. Using RNA immunoprecipitation, we demonstrate that GAPDH-TNF mRNA binding increases when THP-1 monocytes are in a low glycolysis state, and that this binding can be reversed by knocking down GAPDH expression or by increasing glycolysis. We show that reducing glycolysis decreases TNF mRNA association with polysomes. We demonstrate that GAPDH-TNF mRNA binding results in posttranscriptional repression of TNF and that the TNF mRNA 3' untranslated region is sufficient for repression. Finally, after exploring this model in THP-1 cells, we demonstrate this mechanism affects TNF expression in primary human monocytes and macrophages. We conclude that GAPDH-TNF mRNA binding regulates expression of TNF based on cellular metabolic state. We think this mechanism has potentially significant implications for treatment of various immunometabolic conditions, including immune paralysis during septic shock.

Posttranscriptional and Translational Control of Gene Regulation in CD4+ T Cell Subsets.

The immune system is under strict regulatory control to ensure homeostasis of inflammatory responses, lying dormant when not needed but quick to act when called upon. Small changes in gene expression can lead to drastic changes in lineage commitment, cellular function, and immunity. Conventional assessment of these changes centered on the analysis of mRNA levels through a variety of methodologies, including microarrays. However, mRNA synthesis does not always correlate directly to protein synthesis and downstream functional activity. Work conducted in recent years has begun to shed light on the various posttranscriptional changes that occur in response to a dynamic external environment that a given cell type encounters. We provide a critical review of key posttranscriptional mechanisms (i.e., microRNA) and translational mechanisms of regulation of gene expression in the immune system, with a particular emphasis on these regulatory processes in various CD4(+) T cell subsets.

Stress granules and RNA processing bodies are novel autoantibody targets in systemic sclerosis.

BACKGROUND: Autoantibody profiles represent important patient stratification markers in systemic sclerosis (SSc). Here, we performed serum-immunoprecipitations with patient antibodies followed by mass spectrometry (LC-MS/MS) to obtain an unbiased view of all possible autoantibody targets and their associated molecular complexes recognized by SSc. METHODS: HeLa whole cell lysates were immunoprecipitated (IP) using sera of patients with SSc clinically positive for autoantibodies against RNA polymerase III (RNAP3), topoisomerase 1 (TOP1), and centromere proteins (CENP). IP eluates were then analyzed by LC-MS/MS to identify novel proteins and complexes targeted in SSc. Target proteins were examined using a functional interaction network to identify major macromolecular complexes, with direct targets validated by IP-Western blots and immunofluorescence. RESULTS: A wide range of peptides were detected across patients in each clinical autoantibody group. Each group contained peptides representing a broad spectrum of proteins in large macromolecular complexes, with significant overlap between groups. Network analyses revealed significant enrichment for proteins in RNA processing bodies (PB) and cytosolic stress granules (SG) across all SSc subtypes, which were confirmed by both Western blot and immunofluorescence. CONCLUSIONS: While strong reactivity was observed against major SSc autoantigens, such as RNAP3 and TOP1, there was overlap between groups with widespread reactivity seen against multiple proteins. Identification of PB and SG as major targets of the humoral immune response represents a novel SSc autoantigen and suggests a model in which a combination of chronic and acute cellular stresses result in aberrant cell death, leading to autoantibody generation directed against macromolecular nucleic acid-protein complexes.

RNA binding proteins as regulators of immune cell biology.

Sequence-specific RNA binding proteins (RBP) are important regulators of the immune response. RBP modulate gene expression by regulating splicing, polyadenylation, localization, translation and decay of target mRNAs. Increasing evidence suggests that RBP play critical roles in the development, activation and function of lymphocyte populations in the immune system. This review will discuss the post-transcriptional regulation of gene expression by RBP during lymphocyte development, with particular focus on the Tristetraprolin family of RBP.

Arabidopsis TAF15b Localizes to RNA Processing Bodies and Contributes to snc1-Mediated Autoimmunity.

In both animals and plants, messenger (m)RNA export has been shown to contribute to immune response regulation. The Arabidopsis nuclear protein MOS11, along with the nucleoporins MOS3/Nup96/SAR3 and Nup160/SAR1 are components of the mRNA export machinery and contribute to immunity mediated by nucleotide binding leucine-rich repeat immune receptors (NLR). The human MOS11 ortholog CIP29 is part of a small protein complex with three additional members: the RNA helicase DDX39, ALY, and TAF15b. We systematically assessed the biological roles of the Arabidopsis homologs of these proteins in toll interleukin 1 receptor-type NLR (TNL)-mediated immunity using reverse genetics. Although mutations in ALY and DDX39 did not result in obvious defects, taf15b mutation partially suppressed the autoimmune phenotypes of a gain-of-function TNL mutant, snc1. An additive effect on snc1 suppression was observed in mos11-1 taf15b snc1 triple mutant plants, suggesting that MOS11 and TAF15b have independent functions. TAF15b-GFP fusion protein, which fully complemented taf15b mutant phenotypes, localized to nuclei similarly to MOS11. However, it was also targeted to cytosolic granules identified as processing bodies. In addition, we observed no change in SNC1 mRNA levels, whereas less SNC1 protein accumulated in taf15b mutant, suggesting that TAF15b contributes to SNC1 homeostasis through posttranscriptional mechanisms. In summary, this study highlights the importance of posttranscriptional RNA processing mediated by TAF15b in the regulation of TNL-mediated immunity.

Recognition of Specified RNA Modifications by the Innate Immune System.

Microbial nucleic acids have been described as important activators of human innate immune responses by triggering so-called pattern recognition receptors (PRRs) that are expressed on innate immune cells, including plasmacytoid dendritic cells and monocytes. Although host and microbial nucleic acids share pronounced chemical and structural similarities, they significantly differ in their posttranscriptional modification profile, allowing the host to discriminate between self and nonself. In this regard, ribose 2'-O-methylation has been discovered as suppressor of RNA-induced PRR activation. Although 2'-O-methylation occurs with higher frequencies in eukaryotic than in prokaryotic RNA, the immunosuppressive properties of 2'-O-methylated nucleotides may be misused by certain bacteria as immune evasion mechanism. In the course of identifying inhibitory RNA modifications, our groups have synthesized and comparatively analyzed a series of differentially modified RNAs, so-called modivariants, for their immune stimulatory capacities. In this chapter, we will detail the protocols for the design and synthesis of RNA modivariants by molecular cut-and-paste techniques (referred to as molecular surgery) and describe testing of their immune stimulatory properties upon transfection into peripheral blood mononuclear cells.