Adenosine-to-Inosine RNA editing in cancer: molecular mechanisms and downstream targets.

Adenosine-to-Inosine (A-to-I), one of the most prevalent RNA modifications, has recently garnered significant attention. The A-to-I modification actively contributes to biological and pathological processes by affecting the structure and function of various RNA molecules, including double stranded RNA, transfer RNA, microRNA, and viral RNA. Increasing evidence suggests that A-to-I plays a crucial role in the development of human disease, particularly in cancer, and aberrant A-to-I levels are closely associated with tumorigenesis and progression through regulation of the expression of multiple oncogenes and tumor suppressor genes. Currently, the underlying molecular mechanisms of A-to-I modification in cancer are not comprehensively understood. Here, we review the latest advances regarding the A-to-I editing pathways implicated in cancer, describing their biological functions and their connections to the disease.

Prophylactic supplementation with Bifidobacterium infantis or its metabolite inosine attenuates cardiac ischemia/reperfusion injury.

Emerging evidence has demonstrated the profound impact of the gut microbiome on cardiovascular diseases through the production of diverse metabolites. Using an animal model of myocardial ischemia-reperfusion (I/R) injury, we found that the prophylactic administration of a well-known probiotic, Bifidobacterium infantis (B. infantis), exhibited cardioprotective effects in terms of preserving cardiac contractile function and preventing adverse cardiac remodeling following I/R and that these cardioprotective effects were recapitulated by its metabolite inosine. Transcriptomic analysis further revealed that inosine mitigated I/R-induced cardiac inflammation and cell death. Mechanistic investigations elucidated that inosine suppressed the production of pro-inflammatory cytokines and reduced the numbers of dendritic cells and natural killer cells, achieved through the activation of the adenosine A2A receptor (A2AR) that when inhibited abrogated the cardioprotective effects of inosine. Additionally, in vitro studies using C2C12 myoblasts revealed that inosine attenuated cell death by serving as an alternative carbon source for adenosine triphosphate (ATP) generation through the purine salvage pathway when subjected to oxygen-glucose deprivation/reoxygenation that simulated myocardial I/R injury. Likewise, inosine reversed the I/R-induced decrease in ATP levels in mouse hearts. Taken together, our findings indicate that B. infantis or its metabolite inosine exerts cardioprotective effects against I/R by suppressing cardiac inflammation and attenuating cardiac cell death, suggesting prophylactic therapeutic options for acute ischemic cardiac injury.

Roncoroni Re-Visited: The Neuronal Intranuclear Rodlet Comes of Age.

Despite myriad technological advances in neuroscience, the nervous system harbors morphological phenomena that continue to defy explanation. First described by the classical microscopists, including Santiago Ramon y Cajal, at the end of the 19th century, the neuronal intranuclear rodlet (INR) has mystified neurohistologists and microscopists for centuries. In this review article, we will provide an overview of the discovery of the INR as well as the subsequent attempts to elucidate its nature and functional significance. We outline our own studies of this structure over the past three decades, focusing on its elusive nature, its interactions with other nuclear organelles, and on disease-related quantitative changes in Alzheimer's disease. We then describe our somewhat serendipitous discovery that these structures are filamentous aggregates of the nucleotide-synthesizing metabolic enzyme inosine monophosphate dehydrogenase. The filamentation of metabolic enzymes to form mesoscale cellular structures called "rods and rings" or "cytoophidia" (Greek for "cellular snakes") is a recently described phenomenon that remains to be systematically investigated in the nervous system. Thus, this review provides an intriguing historical juxtaposition in neuroscience, inculcating the neuronal INR, once a mere morphological curiosity, into one of the most rapidly evolving fields in contemporary cell biology.

Purine metabolite inosine induced by transforming growth factor­ß promotes epithelial­mesenchymal transition in colorectal cancer.

Transforming growth factor-ß (TGF-ß) signaling pathway serves a pivotal role in the pathogenesis of colorectal cancer (CRC). However, the specific molecular mechanisms by which the TGF-ß signaling pathway regulates CRC are still not fully understood. In the present study, metabolomics and transcriptomics were used to screen for key metabolites and regulatory genes most related to the regulation of the TGF-ß signaling pathway in CRC. Additionally, reverse transcription-quantitative PCR, western blotting and Transwell assays were performed to assess the process of epithelial-mesenchymal transition (EMT). Metabolomics analysis indicated that TGF-ß1 has an impact on purine metabolism, leading to an increase in the purine metabolite inosine. The increase of inosine is essential for facilitating EMT and cell migration in CRC cells. Furthermore, the integrated analysis of metabolomics and transcriptomics data revealed that TGF-ß1 induces the expression of laccase domain-containing 1 (LACC1), an enzyme involved in the regulation of inosine. Knockdown of LACC1 resulted in a reduction of TGF-ß1-induced alterations in inosine levels, EMT and cell migration in CRC cells. The results of the present study suggest that the TGF-ß signaling pathway is involved in the regulation of purine metabolism in CRC through the modulation of LACC1 expression. Furthermore, LACC1 appears to influence EMT and cell migration by elevating the levels of the purine metabolite inosine.

ADR-2 regulates fertility and oocyte fate in C. elegans.

RNA binding proteins play essential roles in coordinating germline gene expression and development in all organisms. Here, we report that loss of ADR-2, a member of the Adenosine DeAminase acting on RNA (ADAR) family of RNA binding proteins and the sole adenosine-to-inosine RNA editing enzyme in C. elegans, can improve fertility in multiple genetic backgrounds. First, we show that loss of RNA editing by ADR-2 restores normal embryo production to subfertile animals that transgenically express a vitellogenin (yolk protein) fusion to green fluorescent protein. Using this phenotype, a high-throughput screen was designed to identify RNA binding proteins that when depleted yield synthetic phenotypes with loss of adr-2. The screen uncovered a genetic interaction between ADR-2 and SQD-1, a member of the heterogenous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins. Microscopy, reproductive assays, and high-throughput sequencing reveal that sqd-1 is essential for the onset of oogenesis and oogenic gene expression in young adult animals, and that loss of adr-2 can counteract the effects of loss of sqd-1 on gene expression and rescue the switch from spermatogenesis to oogenesis. Together, these data demonstrate that ADR-2 can contribute to the suppression of fertility and suggest novel roles for both RNA editing-dependent and independent mechanisms in regulating embryogenesis.

Base preference for inosine 3'-riboendonuclease activity of human endonuclease V: implications for cleavage of poly-A tails containing inosine.

Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.

Insights into ADAR gene complement, expression patterns, and RNA editing landscape in Chlamys farreri.

Adenosine Deaminases Acting on RNA (ADARs) are evolutionarily conserved enzymes known to convert adenosine to inosine in double-stranded RNAs and participate in host-virus interactions. Conducting a meta-analysis of available transcriptome data, we identified and characterised eight ADAR transcripts in Chlamys farreri, a farmed marine scallop susceptible to Acute viral necrosis virus (AVNV) infections and mortality outbreaks. Accordingly, we identified six ADAR genes in the Zhikong scallop genome, revised previous gene annotations, and traced alternative splicing variants. In detail, each ADAR gene encodes a unique combination of functional domains, always including the Adenosine deaminase domain, RNA binding domains and, in one case, two copies of a Z-DNA binding domain. After phylogenetic analysis, five C. farreri ADARs clustered in the ADAR1 clade along with sequences from diverse animal phyla. Gene expression analysis indicated CF051320 as the most expressed ADAR, especially in the eye and male gonad. The other four ADAR1 genes and one ADAR2 gene exhibited variable expression levels, with CF105370 and CF051320 significantly increasing during early scallop development. ADAR-mediated single-base editing, evaluated across adult C. farreri tissues and developmental stages, was mainly detectable in intergenic regions (83 % and 85 %, respectively). Overall, the expression patterns of the six ADAR genes together with the editing and hyper-editing values computed on scallops RNA-seq samples support the adaptive value of ADAR1-mediated editing, particularly in the pre-settling larval stages.

Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus.

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyse the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA modifications in viral RNA genomes.

Transcriptome analysis reveals the role of long noncoding RNAs in specific deposition of inosine monphosphate in Jingyuan chickens.

Inosine monphosphate (IMP) is one of the important indicators for evaluating meat flavor, and long noncoding RNAs (lncRNAs) play an important role in its transcription and post-transcriptional regulation. Currently, there is little information about how lncRNA regulates the specific deposition of IMP in chicken muscle. In this study, we used transcriptome sequencing to analyze the lncRNAs of the breast and leg muscles of the Jingyuan chicken and identified a total of 357 differentially expressed lncRNAs (DELs), of which 158 were up-regulated and 199 were down-regulated. There were 2,203 and 7,377 cis- and trans-regulated target genes of lncRNAs, respectively, and we identified the lncRNA target genes that are involved in NEGF signaling pathway, glycolysis/glucoseogenesis, and biosynthesis of amino acids pathways. Meanwhile, 621 pairs of lncRNA-miRNA-mRNA interaction networks were constructed with target genes involved in purine metabolism, fatty acid metabolism, and biosynthesis of amino acids. Next, three interacting meso-networks gga-miR-1603-LNC_000324-PGM1, gga-miR-1768-LNC_000324-PGM1, and gga-miR-21-LNC_011339-AMPD1 were identified as closely associated with IMP-specific deposition. Both differentially expressed genes (DEGs) PGM1 and AMPD1 were significantly enriched in IMP synthesis and metabolism-related pathways, and participated in the anabolic process of IMP in the form of organic matter synthesis and energy metabolism. This study obtained lncRNAs and target genes affecting IMP-specific deposition in Jingyuan chickens based on transcriptome analysis, which deepened our insight into the role of lncRNAs in chicken meat quality.

Jingyuan chicken is an excellent local chicken breed listed in the Catalogue of Livestock and Poultry Genetic Resources of China. Its unique growing environment has enabled Jingyuan chicken to develop the characteristics of compact meat, unique flavor, and high nutritional value, which makes it the first choice for chicken food. Inosine monophosphate (IMP) is widely recognized as an important indicator for evaluating the flavor of livestock and poultry meat. To mine potential long noncoding RNAs (lncRNAs) and their regulatory IMP-specific deposition interaction networks, we used transcriptome sequencing to identify 357 lncRNAs that were differentially expressed in breast and leg muscles of 180-d-old Jingyuan hens. We screened the key lncRNAs affecting IMP and three lncRNA-miRNA-mRNA regulatory networks by bioinformatics methods. This provides a new approach to studying IMP-specific deposition, improvement of chicken meat flavor, and breed improvement in Jingyuan chickens.

Dichotomous roles of ADAR1 in liver hepatocellular carcinoma and kidney renal cell carcinoma: Unraveling the complex tumor microenvironment and prognostic significance.

BACKGROUND: Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-editing enzyme that significantly impacts cancer progression and various biological processes. The expression of ADAR1 mRNA has been examined in multiple cancer types using The Cancer Genome Atlas (TCGA) dataset, revealing distinct patterns in kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and liver hepatocellular carcinoma (LIHC) compared to normal controls. However, the reasons for these differential expressions remain unclear. METHODS: In this study, we performed RT-PCR and western blotting (WB) to validate ADAR1 expression patterns in clinical tissue samples. Survival analysis and immune microenvironment analysis (including immune score and stromal score) were conducted using TCGA data to determine the specific cell types associated with ADAR1, as well as the key genes in those cell types. The relationship between ADAR1 and specific cell types' key genes was verified by immunohistochemistry (IHC), using clinical liver and kidney cancer samples. RESULTS: Our validation analysis revealed that ADAR1 expression was downregulated in KICH, KIRC, and KIRP, while upregulated in LIHC compared to normal tissues. Notably, a significant correlation was found between ADAR1 mRNA expression and patient prognosis, particularly in KIRC, KIRP, and LIHC. Interestingly, we observed a positive correlation between ADAR1 expression and stromal scores in KIRC, whereas a negative correlation was observed in LIHC. Cell type analysis highlighted distinct relationships between ADAR1 expression and the two stromal cell types, blood endothelial cells (BECs) and lymphatic endothelial cells (LECs), and further determined the signature gene claudin-5 (CLDN5), in KIRC and LIHC. Moreover, ADAR1 was inversely related with CLDN5 in KIRC (n = 26) and LIHC (n = 30) samples, verified via IHC. CONCLUSIONS: ADAR1 plays contrasting roles in LIHC and KIRC, associated with the enrichment of BECs and LECs within tumors. This study sheds light on the significant roles of stromal cells within the complex tumor microenvironment (TME) and provides new insights for future research in tumor immunotherapy and precision medicine.

Codon decoding by orthogonal tRNAs interrogates the in vivo preferences of unmodified adenosine in the wobble position.

The in vivo codon decoding preferences of tRNAs with an authentic adenosine residue at position 34 of the anticodon, the wobble position, are largely unexplored because very few unmodified A34 tRNA genes exist across the three domains of life. The expanded wobble rules suggest that unmodified adenosine pairs most strongly with uracil, modestly with cytosine, and weakly with guanosine and adenosine. Inosine, a modified adenosine, on the other hand, pairs strongly with both uracil and cytosine and to a lesser extent adenosine. Orthogonal pair directed sense codon reassignment experiments offer a tool with which to interrogate the translational activity of A34 tRNAs because the introduced tRNA can be engineered with any anticodon. Our fluorescence-based screen utilizes the absolute requirement of tyrosine at position 66 of superfolder GFP for autocatalytic fluorophore formation. The introduced orthogonal tRNA competes with the endogenous translation machinery to incorporate tyrosine in response to a codon typically assigned another meaning in the genetic code. We evaluated the codon reassignment efficiencies of 15 of the 16 possible orthogonal tRNAs with A34 anticodons. We examined the Sanger sequencing chromatograms for cDNAs from each of the reverse transcribed tRNAs for evidence of inosine modification. Despite several A34 tRNAs decoding closely-related C-ending codons, partial inosine modification was detected for only three species. These experiments employ a single tRNA body with a single attached amino acid to interrogate the behavior of different anticodons in the background of in vivo E. coli translation and greatly expand the set of experimental measurements of the in vivo function of A34 tRNAs in translation. For the most part, unmodified A34 tRNAs largely pair with only U3 codons as the original wobble rules suggest. In instances with GC pairs in the first two codon positions, unmodified A34 tRNAs decode the C- and G-ending codons as well as the expected U-ending codon. These observations support the "two-out-of-three" and "strong and weak" codon hypotheses.

Radioprotective Properties of Riboxin (Inosine) and Indralin under External Irradiation.

A comparative assessment of radioprotective properties of inosine nucleoside (riboxin) and recognized radioprotector indralin was carried out. We analyzed survival of male ICR CD-1 mice weighting 32.2±0.2 g exposed to external X-ray radiation at doses 6.5 and 6.75 Gy and receiving indralin at a dose of 100 or 150 µg/g body weight or riboxin (inosine) at a dose of 100 or 200 µg/g body weight before irradiation. The survival analysis was carried out by the Kaplan-Meier method. The significance was assessed by using the log-rank-test. Inosine showed a significant difference from the irradiated control only at a dose of 100 µg/g body weight at a radiation dose of 6.75 Gy. The survival of animals treated with indralin was significantly higher in comparison with not only the irradiated control group, but also with the groups receiving inosine.

Meat Characteristics, Expression of Myosin Heavy Chain and Metabolism-Related Genes in Thai Native Pigs.

This study investigated the meat quality, expression of myosin heavy chain (MyHC) and metabolism-related genes, ribonucleotides and fatty acids in Longissimus thoracis of Thai native pigs (TNPs) from different geographical regions (GR). Forty-one 9-10-month-old castrated TNPs (BW 60 kg), consisting of 18, 11 and 12 pigs from Northern (NT), Southern (ST) and Northeastern (NE) regions, respectively, were slaughtered. GR did not affect (p > 0.05) the expression of MyHC, phosphoglycerate mutase 1, cytosolic glycerol-3-phosphate dehydrogenase, triosephosphate isomerase 1 and adipocyte fatty acid binding protein genes. The trend of MyHC was MyHC IIx > MyHC IIb > MyHC IIa > MyHC I. The NT loin had higher (p < 0.05) glycogen, C18:2n6, C20:4n6 and cooking loss, lower inosine, inosine monophosphate and hypoxanthine and a shorter sarcomere length than the ST and NE loins. The ST loin had a lower (p < 0.05) a* compared to other loins. Principal component analysis established significant relationships between the TNP and specific meat quality traits. This finding suggests that GR affected the meat quality, ribonucleotides and selected fatty acids in TNPs. These results provide relevant information that can be used to optimize the use of Thai native pork.

An Untargeted Metabolomic Analysis of Lacticaseibacillus (L.) rhamnosus, Lactobacillus (L.) acidophilus, Lactiplantibacillus (L.) plantarum and Limosilactobacillus (L.) reuteri Reveals an Upregulated Production of Inosine from L. rhamnosus.

Lactic acid bacteria are considered an inexhaustible source of bioactive compounds; indeed, products from their metabolism are known to have immunomodulatory and anti-inflammatory activity. Recently, we demonstrated that Cell-Free Supernatants (CFS) obtained from Lactobacillus (L.) acidophilus, Lactiplantibacillus (L.) plantarum, Lacticaseibacillus (L.) rhamnosus, and Limosilactobacillus (L.) reuteri can impair Candida pathogenic potential in an in vitro model of epithelial vaginal infection. This effect could be ascribed to a direct effect of living lactic acid bacteria on Candida virulence and to the production of metabolites that are able to impair fungal virulence. In the present work, stemming from these data, we deepened our knowledge of CFS from these four lactic acid bacteria by performing a metabolomic analysis to better characterize their composition. By using an untargeted metabolomic approach, we detected consistent differences in the metabolites produced by these four different lactic acid bacteria. Interestingly, L. rhamnosus and L. acidophilus showed the most peculiar metabolic profiles. Specifically, after a hierarchical clustering analysis, L. rhamnosus and L. acidophilus showed specific areas of significantly overexpressed metabolites that strongly differed from the same areas in other lactic acid bacteria. From the overexpressed compounds in these areas, inosine from L. rhamnosus returned with the best identification profile. This molecule has been described as having antioxidant, anti-inflammatory, anti-infective, and neuroprotective properties. The biological significance of its overproduction by L. rhamnosus might be important in its probiotic and/or postbiotic activity.

Effects of Dietary Inosine 5'-Monophosphate Supplementation on the Growth Performance and Salinity and Oxidative Stress Resistance of Gibel Carp (Carassius auratus gibelio).

An 88-day feeding trial was conducted to evaluate the effects of dietary inosine 5'-monophosphate (5'-IMP) on the growth performance and salinity and oxidative stress resistance in the juvenile gibel carp CAS III (Carassius auratus gibelio; initial body weight: 7.48 g). Four isonitrogenous and isoenergetic diets containing exogenous 5'-IMP were formulated. P1, P2, P3 and P4 were diets containing 5'-IMP at four concentrations (0, 1, 2 and 4 g kg-1). The four diets were randomly allotted to triplicate tanks in a recirculating system. After the feeding trial, six fish per tank were netted randomly and placed into 12‱ saline water to test their response to salinity stress. The results indicated that the feed conversion rate was enhanced by dietary supplementation with 5'-IMP. The appetite, plasma neuropeptide Y level and feeding rate of the P3 group were lower than those in the control treatment group. Dietary supplementation with 5'-IMP improved the osmoregulatory adaptation of gibel carp under acute salinity stress. Six hours after the salinity stress treatment, in the dietary 5'-IMP treatment group, the plasma cortisol and K+ concentrations were lower and the Na+/K+-ATPase activity was greater than that in the control group. Dietary supplementation with 5'-IMP promoted the expression of the glucocorticoid receptors NKA-α1b and NKCC and retarded the expression of Hsp70 in P4-treated gill filaments and kidneys. Dietary supplementation with 5'-IMP resulted in a stable oxidative-stress-resistant phenotype characterized by increased levels of cellular antioxidants, including SOD, catalase, glutathione peroxidase, glutathione reductase and MPO. The above results of the current study demonstrate that supplementation of 5'-IMP can promote feed utilization and have positive influences on the salinity and oxidative stress resistance of gibel carp.

Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity.

Chimeric antigen receptor (CAR) T cell therapy is hindered in solid tumor treatment due to the immunosuppressive tumor microenvironment and suboptimal T cell persistence. Current strategies do not address nutrient competition in the microenvironment. Hence, we present a metabolic refueling approach using inosine as an alternative fuel. CAR T cells were engineered to express membrane-bound CD26 and cytoplasmic adenosine deaminase 1 (ADA1), converting adenosine to inosine. Autocrine secretion of ADA1 upon CD3/CD26 stimulation activates CAR T cells, improving migration and resistance to transforming growth factor ß1 suppression. Fusion of ADA1 with anti-CD3 scFv further boosts inosine production and minimizes tumor cell feeding. In mouse models of hepatocellular carcinoma and non-small cell lung cancer, metabolically refueled CAR T cells exhibit superior tumor reduction compared to unmodified CAR T cells. Overall, our study highlights the potential of selective inosine refueling to enhance CAR T therapy efficacy against solid tumors.

Symbiotic combination of Akkermansia muciniphila and inosine alleviates alcohol-induced liver injury by modulating gut dysbiosis and immune responses.

Background: Alcoholic liver disease (ALD) is exacerbated by disruptions in intestinal microecology and immune imbalances within the gut-liver axis. The present study assesses the therapeutic potential of combining Akkermansia muciniphila (A. muciniphila) with inosine in alleviating alcohol-induced liver injury. Methods: Male C57BL/6 mice, subjected to a Lieber-DeCarli diet with 5% alcohol for 4 weeks, served as the alcoholic liver injury model. Various analyzes, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), ELISA, immunochemistry, 16S rRNA gene sequencing, and flow cytometry, were employed to evaluate liver injury parameters, intestinal barrier function, microbiota composition, and immune responses. Results: Compared to the model group, the A. muciniphila and inosine groups exhibited significantly decreased alanine aminotransferase, aspartate aminotransferase, and lipopolysaccharide (LPS) levels, reduced hepatic fat deposition and neutrophil infiltration, alleviated oxidative stress and inflammation, and increased expression of intestinal tight junction proteins (Claudin-1, Occludin, and ZO-1). These effects were further pronounced in the A. muciniphila and inosine combination group compared to individual treatments. While alcohol feeding induced intestinal dysbiosis and gut barrier disruption, the combined treatment reduced the abundance of harmful bacteria (Oscillibacter, Escherichia/Shigella, and Alistipes) induced by alcohol consumption, promoting the growth of butyrate-producing bacteria (Akkermansia, Lactobacillus, and Clostridium IV). Flow cytometry revealed that alcohol consumption reduced T regulatory (Treg) populations while increasing those of T-helper (Th) 1 and Th17, which were restored by A. muciniphila combined with inosine treatment. Moreover, A. muciniphila and inosine combination increased the expression levels of intestinal CD39, CD73, and adenosine A2A receptor (A2AR) along with enhanced proportions of CD4+CD39+Treg and CD4+CD73+Treg cells in the liver and spleen. The A2AR antagonist KW6002, blocked the beneficial effects of the A. muciniphila and inosine combination on liver injury in ALD mice. Conclusion: This study reveals that the combination of A. muciniphila and inosine holds promise for ameliorating ALD by enhancing the gut ecosystem, improving intestinal barrier function, upregulating A2AR, CD73, and CD39 expression, modulating Treg cells functionality, and regulating the imbalance of Treg/Th17/Th1 cells, and these beneficial effects are partly A2AR-dependent.

Transketolase promotes MAFLD by limiting inosine-induced mitochondrial activity.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has a global prevalence of about 25% and no approved therapy. Using metabolomic and proteomic analyses, we identified high expression of hepatic transketolase (TKT), a metabolic enzyme of the pentose phosphate pathway, in human and mouse MAFLD. Hyperinsulinemia promoted TKT expression through the insulin receptor-CCAAT/enhancer-binding protein alpha axis. Utilizing liver-specific TKT overexpression and knockout mouse models, we demonstrated that TKT was sufficient and required for MAFLD progression. Further metabolic flux analysis revealed that Tkt deletion increased hepatic inosine levels to activate the protein kinase A-cAMP response element binding protein cascade, promote phosphatidylcholine synthesis, and improve mitochondrial function. Moreover, insulin induced hepatic TKT to limit inosine-dependent mitochondrial activity. Importantly, N-acetylgalactosamine (GalNAc)-siRNA conjugates targeting hepatic TKT showed promising therapeutic effects on mouse MAFLD. Our study uncovers how hyperinsulinemia regulates TKT-orchestrated inosine metabolism and mitochondrial function and provides a novel therapeutic strategy for MAFLD prevention and treatment.

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.

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.