New comparative genomic evidence supporting the proteomic diversification role of A-to-I RNA editing in insects.

Adenosine-to-inosine (A-to-I) RNA editing, resembling A-to-G mutation, confers adaptiveness by increasing proteomic diversity in a temporal-spatial manner. This evolutionary theory named "proteomic diversifying hypothesis" has only partially been tested in very few organisms like Drosophila melanogaster, mainly by observing the positive selection on nonsynonymous editing events. To find additional genome-wide evidences supporting this interesting assumption, we retrieved the genomes of four Drosophila species and collected 20 deep-sequenced transcriptomes of different developmental stages and neuron populations of D. melanogaster. We systematically profiled the RNA editomes in these samples and performed meticulous comparative genomic analyses. Further evidences were found to support the diversifying hypothesis. (1) None of the nonsynonymous editing sites in D. melanogaster had ancestral G-alleles, while the silent editing sites had an unignorable fraction of ancestral G-alleles; (2) Only very few nonsynonymous editing sites in D. melanogaster had corresponding G-alleles derived in the genomes of sibling species, and the fraction of such situation was significantly lower than that of silent editing sites; (3) The few nonsynonymous editing with corresponding G-alleles had significantly more variable editing levels (across samples) than other nonsynonymous editing sites in D. melanogaster. The proteomic diversifying nature of RNA editing in Drosophila excludes the restorative role which favors an ancestral G-allele. The few fixed G-alleles in sibling species might facilitate the adaptation to particular environment and the corresponding nonsynonymous editing in D. melanogaster would introduce stronger advantage of flexible proteomic diversification. With multi-Omics data, our study consolidates the nature of evolutionary significance of A-to-I RNA editing sites in model insects.

Adenosine deaminases that act on RNA, then and now.

In this article, I recount my memories of key experiments that led to my entry into the RNA editing/modification field. I highlight initial observations made by the pioneers in the ADAR field, and how they fit into our current understanding of this family of enzymes. I discuss early mysteries that have now been solved, as well as those that still linger. Finally, I discuss important, outstanding questions and acknowledge my hope for the future of the RNA editing/modification field.

Structural and functional effects of inosine modification in mRNA.

Inosine (I), resulting from the deamination of adenosine (A), is a prominent modification in the human transcriptome. The enzymes responsible for the conversion of adenosine to inosine in human mRNAs are the ADARs (adenosine deaminases acting on RNA). Inosine modification introduces a layer of complexity to mRNA processing and function, as it can impact various aspects of RNA biology, including mRNA stability, splicing, translation, and protein binding. The relevance of this process is emphasized in the growing number of human disorders associated with dysregulated A-to-I editing pathways. Here, we describe the impact of the A-to-I conversion on the structure and stability of duplex RNA and on the consequences of this modification at different locations in mRNAs. Furthermore, we highlight specific open questions regarding the interplay between inosine formation in duplex RNA and the innate immune response.

The first A-to-I RNA editome of hemipteran species Coridius chinensis reveals overrepresented recoding and prevalent intron editing in early-diverging insects.

BACKGROUND: Metazoan adenosine-to-inosine (A-to-I) RNA editing resembles A-to-G mutation and increases proteomic diversity in a temporal-spatial manner, allowing organisms adapting to changeable environment. The RNA editomes in many major animal clades remain unexplored, hampering the understanding on the evolution and adaptation of this essential post-transcriptional modification. METHODS: We assembled the chromosome-level genome of Coridius chinensis belonging to Hemiptera, the fifth largest insect order where RNA editing has not been studied yet. We generated ten head RNA-Seq libraries with DNA-Seq from the matched individuals. RESULTS: We identified thousands of high-confidence RNA editing sites in C. chinensis. Overrepresentation of nonsynonymous editing was observed, but conserved recoding across different orders was very rare. Under cold stress, the global editing efficiency was down-regulated and the general transcriptional processes were shut down. Nevertheless, we found an interesting site with "conserved editing but non-conserved recoding" in potassium channel Shab which was significantly up-regulated in cold, serving as a candidate functional site in response to temperature stress. CONCLUSIONS: RNA editing in C. chinensis largely recodes the proteome. The first RNA editome in Hemiptera indicates independent origin of beneficial recoding during insect evolution, which advances our understanding on the evolution, conservation, and adaptation of RNA editing.

Unlocking CAR T cell potential: Inosine-induced stemness and enhanced potency.

Adenosine (Ado) drives immune suppression in the tumor microenvironment. In this issue of Cancer Cell, Klysz et al. investigate Ado-mediated immunosuppression. Overexpression of Ado deaminase (ADA-OE), metabolizing Ado to inosine (INO), induces stemness and improves CAR T cell functionality. Likewise, exposure to INO enhances CAR T cells' function and induces stemness features.

Inosine induces stemness features in CAR-T cells and enhances potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

Decoding the language of immunity.

Optimized transfer RNA (tRNA) codon use can speed up antibody generation.

Narrowing down the candidates of beneficial A-to-I RNA editing by comparing the recoding sites with uneditable counterparts.

Adar-mediated adenosine-to-inosine (A-to-I) RNA editing mainly occurs in nucleus and diversifies the transcriptome in a flexible manner. It has been a challenging task to identify beneficial editing sites from the sea of total editing events. The functional Ser>Gly auto-recoding site in insect Adar gene has uneditable Ser codons in ancestral nodes, indicating the selective advantage to having an editable status. Here, we extended this case study to more metazoan species, and also looked for all Drosophila recoding events with potential uneditable synonymous codons. Interestingly, in D. melanogaster, the abundant nonsynonymous editing is enriched in the codons that have uneditable counterparts, but the Adar Ser>Gly case suggests that the editable orthologous codons in other species are not necessarily edited. The use of editable versus ancestral uneditable codon is a smart way to infer the selective advantage of RNA editing, and priority might be given to these editing sites for functional studies due to the feasibility to construct an uneditable allele. Our study proposes an idea to narrow down the candidates of beneficial recoding sites. Meanwhile, we stress that the matched transcriptomes are needed to verify the conservation of editing events during evolution.

GDF15 Protects Insulin-Producing Beta Cells against Pro-Inflammatory Cytokines and Metabolic Stress via Increased Deamination of Intracellular Adenosine.

It has been proposed that antidiabetic drugs, such as metformin and imatinib, at least in part, promote improved glucose tolerance in type 2 diabetic patients via increased production of the inflammatory cytokine GDF15. This is supported by studies, performed in rodent cell lines and mouse models, in which the addition or production of GDF15 improved beta-cell function and survival. The aim of the present study was to determine whether human beta cells produce GDF15 in response to antidiabetic drugs and, if so, to further elucidate the mechanisms by which GDF15 modulates the function and survival of such cells. The effects and expression of GDF15 were analyzed in human insulin-producing EndoC-betaH1 cells and human islets. We observed that alpha and beta cells exhibit considerable heterogeneity in GDF15 immuno-positivity. The predominant form of GDF15 present in islet and EndoC-betaH1 cells was pro-GDF15. Imatinib, but not metformin, increased pro-GDF15 levels in EndoC-betaH1 cells. Under basal conditions, exogenous GDF15 increased human islet oxygen consumption rates. In EndoC-betaH1 cells and human islets, exogenous GDF15 partially ameliorated cytokine- or palmitate + high-glucose-induced loss of function and viability. GDF15-induced cell survival was paralleled by increased inosine levels, suggesting a more efficient disposal of intracellular adenosine. Knockdown of adenosine deaminase, the enzyme that converts adenosine to inosine, resulted in lowered inosine levels and loss of protection against cytokine- or palmitate + high-glucose-induced cell death. It is concluded that imatinib-induced GDF15 production may protect human beta cells partially against inflammatory and metabolic stress. Furthermore, it is possible that the GDF15-mediated activation of adenosine deaminase and the increased disposal of intracellular adenosine participate in protection against beta-cell death.

Purine nucleoside phosphorylase inhibition is an effective approach for the treatment of chemical hemorrhagic cystitis.

Hemorrhagic cystitis may be induced by infection, radiation therapy, or medications or may be idiopathic. Along with hemorrhagic features, symptoms include urinary urgency and frequency, dysuria (painful urination), and visceral pain. Cystitis-induced visceral pain is one of the most challenging types of pain to treat, and an effective treatment would address a major unmet medical need. We assessed the efficacy of a purine nucleoside phosphorylase inhibitor, 8-aminoguanine (8-AG), for the treatment of hemorrhagic/ulcerative cystitis. Lower urinary tract (LUT) function and structure were assessed in adult Sprague-Dawley rats, treated chronically with cyclophosphamide (CYP; sacrificed day 8) and randomized to daily oral treatment with 8-AG (begun 14 days prior to CYP induction) or its vehicle. CYP-treated rats exhibited multiple abnormalities, including increased urinary frequency and neural mechanosensitivity, reduced bladder levels of inosine, urothelial inflammation/damage, and activation of spinal cord microglia, which is associated with pain hypersensitivity. 8-AG treatment of CYP-treated rats normalized all observed histological, structural, biochemical, and physiological abnormalities. In cystitis 8-AG improved function and reduced both pain and inflammation likely by increasing inosine, a tissue-protective purine metabolite. These findings demonstrate that 8-AG has translational potential for reducing pain and preventing bladder damage in cystitis-associated LUT dysfunctions.

Antibody production relies on the tRNA inosine wobble modification to meet biased codon demand.

Antibodies are produced at high rates to provide immunoprotection, which puts pressure on the B cell translational machinery. Here, we identified a pattern of codon usage conserved across antibody genes. One feature thereof is the hyperutilization of codons that lack genome-encoded Watson-Crick transfer RNAs (tRNAs), instead relying on the posttranscriptional tRNA modification inosine (I34), which expands the decoding capacity of specific tRNAs through wobbling. Antibody-secreting cells had increased I34 levels and were more reliant on I34 for protein production than naïve B cells. Furthermore, antibody I34-dependent codon usage may influence B cell passage through regulatory checkpoints. Our work elucidates the interface between the tRNA pool and protein production in the immune system and has implications for the design and selection of antibodies for vaccines and therapeutics.

Inosine-Induced Base Pairing Diversity during Reverse Transcription.

A-to-I editing catalyzed by adenosine deaminase acting on RNAs impacts numerous physiological and biochemical processes that are essential for cellular functions and is a big contributor to the infectivity of certain RNA viruses. The outcome of this deamination leads to changes in the eukaryotic transcriptome functionally resembling A-G transitions since inosine preferentially pairs with cytosine. Moreover, hyper-editing or multiple A to G transitions in clusters were detected in measles virus. Inosine modifications either directly on viral RNA or on cellular RNA can have antiviral or pro-viral repercussions. While many of the significant roles of inosine in cellular RNAs are well understood, the effects of hyper-editing of A to I on viral polymerase activity during RNA replication remain elusive. Moreover, biological strategies such as molecular cloning and RNA-seq for transcriptomic interrogation rely on RT-polymerase chain reaction with little to no emphasis placed on the first step, reverse transcription, which may reshape the sequencing results when hypermodification is present. In this study, we systematically explore the influence of inosine modification, varying the number and position of inosines, on decoding outcomes using three different reverse transcriptases (RTs) followed by standard Sanger sequencing. We find that inosine alone or in clusters can differentially affect the RT activity. To gain structural insights into the accommodation of inosine in the polymerase site of HIV-1 reverse transcriptase (HIV-1-RT) and how this structural context affects the base pairing rules for inosine, we performed molecular dynamics simulations of the HIV-1-RT. The simulations highlight the importance of the protein-nucleotide interaction as a critical factor in deciphering the base pairing behavior of inosine clusters. This effort sets the groundwork for decrypting the physiological significance of inosine and linking the fidelity of reverse transcriptase and the possible diverse transcription outcomes of cellular RNAs and/or viral RNAs where hyper-edited inosines are present in the transcripts.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

Dissection of integrated readout reveals the structural thermodynamics of DNA selection by transcription factors.

Nucleobases such as inosine have been extensively utilized to map direct contacts by proteins in the DNA groove. Their deployment as targeted probes of dynamics and hydration, which are dominant thermodynamic drivers of affinity and specificity, has been limited by a paucity of suitable experimental models. We report a joint crystallographic, thermodynamic, and computational study of the bidentate complex of the arginine side chain with a Watson-Crick guanine (Arg×GC), a highly specific configuration adopted by major transcription factors throughout the eukaryotic branches in the Tree of Life. Using the ETS-family factor PU.1 as a high-resolution structural framework, inosine substitution for guanine resulted in a sharp dissection of conformational dynamics and hydration and elucidated their role in the DNA specificity of PU.1. Our work suggests an under-exploited utility of modified nucleobases in untangling the structural thermodynamics of interactions, such as the Arg×GC motif, where direct and indirect readout are tightly integrated.

Purine Metabolic Pathway Alterations and Serum Urate Changes after Oral Inosine Loading in Male Chinese Volunteers.

BACKGROUND: Oral inosine loading is a new method to evaluate the effects of purine on urate metabolism. However, individuals respond differently to acute purine intake, and the effects on the metabolism of other purines remain to be explored. METHODS: 35 male participants are recruited. Participants received 500 mg of inosine orally after an overnight fast, and blood and urine samples are collected before and at various time points over 180 min after inosine administration. RESULTS: The serum urate concentration is significantly different between the hyperuricemia (n = 14) and non-hyperuricemia (n = 16) groups before inosine intake, but there is no in urate change after inosine intake. When grouped according to the baseline estimated glomerular filtration rate (eGFR), the increase in urate level in the high-eGFR group is significantly higher than that in the low-eGFR group (p  =  0.047). The high-eGFR group showed higher levels of serum xanthine and xanthine oxidase (XOD), the key enzyme in urate synthesis, after inosine loading (p < 0.01). CONCLUSIONS: The increase in urate level is positively related to eGFR after oral acute inosine administration, which may have been due to a higher level of XOD.

Design, synthesis, and cell-based in vitro assay of deoxyinosine-mixed SATE-dCDN prodrugs that activate all common STING variants.

Developing therapeutic strategies to modulate the activity of all prevalent variants (wild-type, HAQ, R232H, AQ, and R293Q) of the stimulator of interferon genes (STING) is still of great interest to treating immune-related diseases. Herein, we synthesized six novel deoxyinosine-mixed deoxyribose cyclic dinucleotide prodrugs (SATE-dCDN) including a combination of hypoxanthine and other bases (A, U, C, T, and G) for a cell-based in vitro assay. The HPLC assay indicated that deoxyinosine-mixed SATE (S-acylthioalkyl ester)-dCDN prodrugs retained high serum stability. The IRF3-responsive luciferase assay in THP1-Lucia cells showed that the activity of the prodrugs with purine bases (SATE-3',3'-c-di-dIMP, SATE-3',3'-c-di-dIdAMP, and SATE-3',3'-c-di-dIdGMP) was higher than that of the prodrugs with pyrimidine bases (SATE-3',3'-c-di-dIdUMP, SATE-3',3'-c-di-dIdTMP, and SATE-3',3'-c-di-dIdCMP), among which prodrug 14a (SATE-3',3'-c-di-dIdAMP) with hypoxanthine and adenine bases exhibited the highest activity with an EC50 value of 0.046 µM. The IRF3 responsive dual-luciferase reporter assay in HEK293T cells transfected with plasmids expressing different STING variants further showed that prodrug 14a could activate all five most common hSTING variants, including the refractory hSTINGR232H and hSTINGQ variants. Furthermore, prodrug 14a also induced the production of the highest levels of mRNA of IFN-ß, CXCL10, IL-6 and TNF-α through STING-dependent IRF and NF-κB signaling pathways in THP-1 cells. These results suggested that the combination of deoxyinosine with a SATE-dCDN prodrug could modulate the broad-spectrum activity of all common STING variants.

Development of an Inosine Hyperproducer from Bacillus licheniformis by Systems Metabolic Engineering.

Inosine is widely used in food, chemical, and medicine. This study developed Bacillus licheniformis into an inosine hyperproducer through systems metabolic engineering. First, purine metabolism was activated by deleting inhibitors PurR and YabJ and overexpressing the pur operon. Then, the 5-phosphoribosyl-1-pyrophosphate (PRPP) supply was increased by optimizing the glucose transport system and pentose phosphate pathway, increasing the inosine titer by 97% and decreasing the titers of byproducts by 36%. Next, to prevent the degradation of inosine, genes deoD and pupG coding purine nucleoside phosphorylase were deleted, accumulating 0.91 g/L inosine in the culture medium. Additionally, the downregulation of adenosine 5'-monophosphate (AMP) synthesis pathway increased the inosine titer by 409%. Importantly, enhancing the glycine and aspartate supply increased the inosine titer by 298%. Finally, the guanosine synthesis pathway was blocked, leading to strain IR-8-2 producing 27.41 g/L inosine with a 0.46 g inosine/g glucose yield and a 0.38 g/(L·h) productivity in a shake flask.

Recent Advances in Adenosine-to-Inosine RNA Editing in Cancer.

RNA epigenetics, or epitranscriptome, is a growing group of RNA modifications historically classified into two categories: RNA editing and RNA modification. RNA editing is usually understood as post-transcriptional RNA processing (except capping, splicing and polyadenylation) that changes the RNA nucleotide sequence encoded by the genome. This processing can be achieved through the insertion or deletion of nucleotides or deamination of nucleobases, generating either standard nucleotides such as uridine (U) or the rare nucleotide inosine (I). Adenosine-to-inosine (A-to-I) RNA editing is the most prevalent type of RNA modification in mammals and is catalyzed by adenosine deaminase acting on the RNA (ADAR) family of enzymes that recognize double-stranded RNAs (dsRNAs). Inosine mimics guanosine (G) in base pairing with cytidine (C), thereby A-to-I RNA editing alters dsRNA secondary structure. Inosine is also recognized as guanosine by the splicing and translation machineries, resulting in mRNA alternative splicing and protein recoding. Therefore, A-to-I RNA editing is an important mechanism that causes and regulates "RNA mutations" in both normal physiology and diseases including cancer. In this chapter, we reviewed current paradigms and developments in the field of A-to-I RNA editing in the context of cancer.

Identification and Interpretation of A-to-I RNA Editing Events in Insect Transcriptomes.

Adenosine-to-inosine (A-to-I) RNA editing is the most prevalent RNA modification in the nervous systems of metazoans. To study the biological significance of RNA editing, we first have to accurately identify these editing events from the transcriptome. The genome-wide identification of RNA editing sites remains a challenging task. In this review, we will first introduce the occurrence, regulation, and importance of A-to-I RNA editing and then describe the established bioinformatic procedures and difficulties in the accurate identification of these sit esespecially in small sized non-model insects. In brief, (1) to obtain an accurate profile of RNA editing sites, a transcriptome coupled with the DNA resequencing of a matched sample is favorable; (2) the single-cell sequencing technique is ready to be applied to RNA editing studies, but there are a few limitations to overcome; (3) during mapping and variant calling steps, various issues, like mapping and base quality, soft-clipping, and the positions of mismatches on reads, should be carefully considered; (4) Sanger sequencing of both RNA and the matched DNA is the best verification of RNA editing sites, but other auxiliary evidence, like the nonsynonymous-to-synonymous ratio or the linkage information, is also helpful for judging the reliability of editing sites. We have systematically reviewed the understanding of the biological significance of RNA editing and summarized the methodology for identifying such editing events. We also raised several promising aspects and challenges in this field. With insightful perspectives on both scientific and technical issues, our review will benefit the researchers in the broader RNA editing community.

Nanogel Particles Based on Modified Nucleosides and Oligosaccharides as Advanced Delivery System.

This work addresses the challenge of delivering bioactive molecules by designing biocompatible nanogel particles (NGPs) utilizing rationally modified nature-sourced building blocks: capryl-oligochitosan and oxidized inosine. Capryl substituents endowed the resultant NGPs with membrane-penetration capabilities, while purine-containing inosine allowed H-bond/π-π/π-cation interactions. The prepared NGPs were complexed with carboxyfluorescein-labeled single-stranded oligonucleotide (FAM-oligo) and DsRed-encoding plasmid DNA. The successful delivery of FAM-oligo to the cell cytoplasm of the Nicotiana benthamiana plant was observed. Alexa 555-labeled bovine serum albumin (Alexa 555-BSA) was also efficiently encapsulated and delivered to the plant. In addition to delivering FAM-oligo and Alexa 555-BSA separately, NGPs also successfully co-delivered both biomolecules to the plant. Finally, NGPs successfully encapsulated the drug amphotericin B and reduced its toxicity while maintaining its efficacy. The presented findings suggest that NGPs may become a promising platform for the advanced delivery of bioactive molecules in various applications.