An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation.

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

Structural perspectives on adenosine to inosine RNA editing by ADARs.

Adenosine deaminases acting on RNA (ADARs) are enzymes that catalyze the hydrolytic deamination of adenosine to inosine. The editing feature of ADARs has garnered much attention as a therapeutic tool to repurpose ADARs to correct disease-causing mutations at the mRNA level in a technique called site-directed RNA editing (SDRE). Administering a short guide RNA oligonucleotide that hybridizes to a mutant sequence forms the requisite dsRNA substrate, directing ADARs to edit the desired adenosine. However, much is still unknown about ADARs' selectivity and sequence-specific effects on editing. Atomic-resolution structures can help provide additional insight to ADARs' selectivity and lead to novel guide RNA designs. Indeed, recent structures of ADAR domains have expanded our understanding on RNA binding and the base-flipping catalytic mechanism. These efforts have enabled the rational design of improved ADAR guide strands and advanced the therapeutic potential of the SDRE approach. While no full-length structure of any ADAR is known, this review presents an exposition of the structural basis for function of the different ADAR domains, focusing on human ADAR2. Key insights are extrapolated to human ADAR1, which is of substantial interest because of its widespread expression in most human tissues.

Pilot-Scale Screening of Clinically Approved Drugs to Identify Uridine Insertion/Deletion RNA Editing Inhibitors in Trypanosoma brucei.

RNA editing pathway is a validated target in kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.) that cause severe diseases in humans and livestock. An essential large protein complex, the editosome, mediates uridine insertion and deletion in RNA editing through a stepwise process. This study details the discovery of editosome inhibitors by screening a library of widely used human drugs using our previously developed in vitro biochemical Ribozyme Insertion Deletion Editing (RIDE) assay. Subsequent studies on the mode of action of the identified hits and hit expansion efforts unveiled compounds that interfere with RNA-editosome interactions and novel ligase inhibitors with IC50 values in the low micromolar range. Docking studies on the ligase demonstrated similar binding characteristics for ATP and our novel epigallocatechin gallate inhibitor. The inhibitors demonstrated potent trypanocidal activity and are promising candidates for drug repurposing due to their lack of cytotoxic effects. Further studies are necessary to validate these targets using more definitive gene-editing techniques and to enhance the safety profile.

Identification of prognostic RNA editing profiles for clear cell renal carcinoma.

Objective: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer and currently lacks effective biomarkers. This research aims to analyze and identify RNA editing profile associated with ccRCC prognosis through bioinformatics and in vitro experiments. Methods: Transcriptome data and clinical information for ccRCC were retrieved from the TCGA database, and RNA editing files were obtained from the Synapse database. Prognostic models were screened, developed, and assessed using consistency index analysis and independent prognostic analysis, etc. Internal validation models were also constructed for further evaluation. Differential genes were investigated using GO, KEGG, and GSEA enrichment analyses. Furthermore, qPCR was performed to determine gene expression in human renal tubular epithelial cells HK-2 and ccRCC cells A-498, 786-O, and Caki-2. Results: An RNA editing-based risk score, that effectively distinguishes between high and low-risk populations, has been identified. It includes CHD3| chr17:7815229, MYO19| chr17:34853704, OIP5-AS1| chr15:41590962, MRI1| chr19:13883962, GBP4| chr1:89649327, APOL1| chr22:36662830, FCF1| chr14:75203040 edited sites or genes and could serve as an independent prognostic factor for ccRCC patients. qPCR results showed significant up-regulation of CHD3, MYO19, MRI1, APOL1, and FCF1 in A-498, 786-O, and Caki-2 cells, while the expression of OIP5-AS1 and GBP4 was significantly down-regulated. Conclusion: RNA editing site-based prognostic models are valuable in differentiating between high and low-risk populations. The seven identified RNA editing sites may be utilized as potential biomarkers for ccRCC.

Adaptive evolution of A-to-I auto-editing site in Adar of eusocial insects.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is a co-/post-transcriptional modification introducing A-to-G variations in RNAs. There is extensive discussion on whether the flexibility of RNA editing exerts a proteomic diversification role, or it just acts like hardwired mutations to correct the genomic allele. Eusocial insects evolved the ability to generate phenotypically differentiated individuals with the same genome, indicating the involvement of epigenetic/transcriptomic regulation. METHODS: We obtained the genomes of 104 Hymenoptera insects and the transcriptomes of representative species. Comparative genomic analysis was performed to parse the evolutionary trajectory of a regulatory Ile > Met auto-recoding site in Adar gene. RESULTS: At genome level, the pre-editing Ile codon is conserved across a node containing all eusocial hymenopterans. At RNA level, the editing events are confirmed in representative species and shows considerable condition-specificity. Compared to random expectation, the editable Ile codon avoids genomic substitutions to Met or to uneditable Ile codons, but does not avoid mutations to other unrelated amino acids. CONCLUSIONS: The flexibility of Adar auto-recoding site in Hymenoptera is selectively maintained, supporting the flexible RNA editing hypothesis. We proposed a new angle to view the adaptation of RNA editing, providing another layer to explain the great phenotypical plasticity of eusocial insects.

A comprehensive atlas of pig RNA editome across 23 tissues reveals RNA editing affecting interaction mRNA-miRNAs.

RNA editing is a co-transcriptional/post-transcriptional modification that is mediated by the ADAR enzyme family. Profiling of RNA editing is very limited in pigs. In this study, we collated 3813 RNA-seq data from the public repositories across 23 tissues and carried out comprehensive profiling of RNA editing in pigs. In total, 127,927 A-to-I RNA editing sites were detected. Our analysis showed that 98.2% of RNA editing sites were located within repeat regions, primarily within the pig-specific SINE retrotransposon PRE-1/Pre0_SS elements. Subsequently, we focused on analyzing specific editing sites (SESs) in skeletal muscle tissues. Functional enrichment analyses suggested that they were enriched in signaling pathways associated with muscle cell differentiation, including DMD, MYOD1 and CAV1 genes. Furthermore, we discovered that RNA editing event in the 3`UTR of CFLAR mRNA influenced miR-708-5p binding in this region. In this study, the panoramic RNA editing landscape of different tissues of pigs was systematically mapped, and RNA editing sites and genes involved in muscle cell differentiation were identified. In summary, we identified modifications to pig RNA editing sites and provided candidate targets for further validation.

Hippocampal adenosine-to-inosine RNA editing in sepsis: dynamic changes and influencing factors.

Sepsis-associated encephalopathy is a diffuse brain dysfunction secondary to infection. It has been established that factors such as age and sex can significantly contribute to the development of sepsis-associated encephalopathy. Our recent study implicated a possible link between adenosine-to-inosine RNA editing and sepsis-associated encephalopathy, yet the dynamics of adenosine-to-inosine RNA editing during sepsis-associated encephalopathy and how it could be influenced by factors such as age, sex and antidepressants remain uninvestigated. Our current study analysed and validated transcriptome-wide changes in adenosine-to-inosine RNA editing in the hippocampus of different septic mouse models. Seventy-four sites in 64 genes showed significant differential RNA editing over time in septic mice induced by caecal ligation and perforation. The differential RNA editing might contribute to the RNA expression regulation of the edited genes, with 42.2% differentially expressed. These differentially edited genes, especially those with missense editing, such as glutamate receptor, ionotropic, kainate 2 (Grik2, p.M620V), filamin A (Flna, p.S2331G) and capicua transcriptional repressor (Cic, p.E2270G), were mainly involved in abnormal social behaviour and neurodevelopmental and psychiatric disorders. Significant effects of age and sex were also observed on sepsis-associated RNA editing. Further comparison highlighted 40 common differential RNA editing sites that caecal ligation and perforation-induced and lipopolysaccharide-induced septic mouse models shared. Interestingly, these findings demonstrate temporal dynamics of adenosine-to-inosine RNA editing in the mouse hippocampus during sepsis, add to the understanding of age and sex differences in the disease and underscore the role of the epigenetic process in sepsis-associated encephalopathy.

Chloroplast genomes of Eriobotrya elliptica and an unknown wild loquat "YN-1".

The chloroplast genomes of wild loquat can help to determine their place in the history of evolution. Here, we sequenced and assembled two novel wild loquat's chloroplast genomes, one is Eriobotrya elliptica, and the other is an unidentified wild loquat, which we named "YN-1". Their sizes are 159,471 bp and 159,399 bp, respectively. We also assembled a cultivated loquat named 'JFZ', its chloroplast genome size is 159,156 bp. A comparative study was conducted with six distinct species of loquats, including five wild loquats and one cultivated loquat. The results showed that both E. elliptica and "YN-1" have 127 genes, one gene more than E. fragrans, which is psbK. Regions trnF-GAA-ndhJ, petG-trnP-UGG, and rpl32-trnL-UAG were found to exhibit high variability. It was discovered that there was a positive selection on rpl22 and rps12. RNA editing analysis found several chilling stress-specific RNA editing sites, especially in rpl2 gene. Phylogenetic analysis results showed that "YN-1" is closely related to E. elliptica, E. obovata and E. henryi.

RBPs: an RNA editor's choice.

RNA-binding proteins (RBPs) play a key role in gene expression and post-transcriptional RNA regulation. As integral components of ribonucleoprotein complexes, RBPs are susceptible to genomic and RNA Editing derived amino acid substitutions, impacting functional interactions. This article explores the prevalent RNA Editing of RBPs, unravelling the complex interplay between RBPs and RNA Editing events. Emphasis is placed on their influence on single amino acid variants (SAAVs) and implications for disease development. The role of Proteogenomics in identifying SAAVs is briefly discussed, offering insights into the RBP landscape. RNA Editing within RBPs emerges as a promising target for precision medicine, reshaping our understanding of genetic and epigenetic variations in health and disease.

Correlations of LncRNA HNF4A-AS1 Alternative Transcripts with Liver Diseases and Drug Metabolism.

Hepatocyte nuclear factor 4 alpha antisense 1 (HNF4A-AS1) is a long non-coding RNA (lncRNA) gene physically located next to the transcription factor HNF4A gene in the human genome. Its transcription products have been reported to inhibit the progression of hepatocellular carcinoma (HCC) and negatively regulate the expression of cytochrome P450s (CYPs), including CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4. By altering CYP expression, lncRNA HNF4A-AS1 also contributes to the susceptibility of drug-induced liver injury. Thus, HNF4A-AS1 lncRNA is a promising target for controlling HCC and modulating drug metabolism. However, HNF4A-AS1 has 4 annotated alternative transcripts in the human genome browsers, and it is unclear which transcripts the siRNAs or shRNAs used in the previous studies are silenced and which transcripts should be used as the target. In this study, 4 annotated and 2 newly identified transcripts were confirmed. These 6 transcripts showed different expression levels in different liver disease conditions, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and obesity. The expression patterns of all HNF4A-AS1 transcripts were further investigated in liver cell growth from human embryonic stem cells to matured hepatocyte-like cells, HepaRG differentiation, and exposure to rifampicin treatment. Several HNF4A-AS1 transcripts highly displayed correlations with these situations. In addition, some of the HNF4A-AS1 transcripts also showed a strong correlation with CYP3A4 during HepaRG maturation and rifampicin exposure. Our findings provide valuable insights into the specific roles of HNF4A-AS1 transcripts, paving the way for more targeted therapeutic strategies for liver diseases and drug metabolism. Significance Statement This study explores the alternative transcripts of HNF4A-AS1, showing how their expression changes in different biological conditions, from various liver diseases to the growth and differentiation of hepatocytes, and drug metabolism. The generated knowledge is essential for understanding the independent roles of different transcripts from the same lncRNA in different liver diseases and drug metabolism situations.

Dynamic A-to-I RNA editing during acute neuroinflammation in sepsis-associated encephalopathy.

Introduction: The activation of cerebral endothelial cells (CECs) has recently been reported to be the earliest acute neuroinflammation event in the CNS during sepsis-associated encephalopathy (SAE). Importantly, adenosine-to-inosine (A-to-I) RNA editing mediated by ADARs has been associated with SAE, yet its role in acute neuroinflammation in SAE remains unclear. Methods: Our current study systematically analyzed A-to-I RNA editing in cerebral vessels, cerebral endothelial cells (CECs), and microglia sampled during acute neuroinflammation after treatment in a lipopolysaccharide (LPS)-induced SAE mouse model. Results: Our results showed dynamic A-to-I RNA editing activity changes in cerebral vessels during acute neuroinflammation. Differential A-to-I RNA editing (DRE) associated with acute neuroinflammation were identified in these tissue or cells, especially missense editing events such as S367G in antizyme inhibitor 1 (Azin1) and editing events in lincRNAs such as maternally expressed gene 3 (Meg3), AW112010, and macrophage M2 polarization regulator (Mm2pr). Importantly, geranylgeranyl diphosphate synthase 1 (Ggps1) and another three genes were differentially edited across cerebral vessels, CECs, and microglia. Notably, Spearman correlation analysis also revealed dramatic time-dependent DRE during acute neuroinflammation, especially in GTP cyclohydrolase1 (Gch1) and non-coding RNA activated by DNA damage (Norad), both with the editing level positively correlated with both post-LPS treatment time and edited gene expression in cerebral vessels and CECs. Discussion: The findings in our current study demonstrate substantial A-to-I RNA editing changes during acute neuroinflammation in SAE, underlining its potential role in the disease.

The de novo assembly and characterization of the complete mitochondrial genome of bottle gourd (Lagenaria siceraria) reveals the presence of homologous conformations produced by repeat-mediated recombination.

Introduction: Bottle gourd is an annual herbaceous plant that not only has high nutritional value and many medicinal applications but is also used as a rootstock for the grafting of cucurbit crops such as watermelon, cucumber and melon. Organellar genomes provide valuable resources for genetic breeding. Methods: A hybrid strategy with Illumina and Oxford Nanopore Technology sequencing data was used to assemble bottle gourd mitochondrial and chloroplast genomes. Results: The length of the bottle gourd mitochondrial genome was 357547 bp, and that of the chloroplast genome was 157121 bp. These genomes had 27 homologous fragments, accounting for 6.50% of the total length of the bottle gourd mitochondrial genome. In the mitochondrial genome, 101 simple sequence repeats (SSRs) and 10 tandem repeats were identified. Moreover, 1 pair of repeats was shown to mediate homologous recombination into 1 major conformation and 1 minor conformation. The existence of these conformations was verified via PCR amplification and Sanger sequencing. Evolutionary analysis revealed that the mitochondrial genome sequence of bottle gourd was highly conserved. Furthermore, collinearity analysis revealed many rearrangements between the homologous fragments of Cucurbita and its relatives. The Ka/Ks values for most genes were between 0.3~0.9, which means that most of the genes in the bottle gourd mitochondrial genome are under purifying selection. We also identified a total of 589 potential RNA editing sites on 38 mitochondrial protein-coding genes (PCGs) on the basis of long noncoding RNA (lncRNA)-seq data. The RNA editing sites of nad1-2, nad4L-2, atp6-718, atp9-223 and rps10-391 were successfully verified via PCR amplification and Sanger sequencing. Conclusion: In conclusion, we assembled and annotated bottle gourd mitochondrial and chloroplast genomes to provide a theoretical basis for similar organelle genomic studies.

Genetics of cell-type-specific post-transcriptional gene regulation during human neurogenesis.

The function of some genetic variants associated with brain-relevant traits has been explained through colocalization with expression quantitative trait loci (eQTL) conducted in bulk postmortem adult brain tissue. However, many brain-trait associated loci have unknown cellular or molecular function. These genetic variants may exert context-specific function on different molecular phenotypes including post-transcriptional changes. Here, we identified genetic regulation of RNA editing and alternative polyadenylation (APA) within a cell-type-specific population of human neural progenitors and neurons. More RNA editing and isoforms utilizing longer polyadenylation sequences were observed in neurons, likely due to higher expression of genes encoding the proteins mediating these post-transcriptional events. We also detected hundreds of cell-type-specific editing quantitative trait loci (edQTLs) and alternative polyadenylation QTLs (apaQTLs). We found colocalizations of a neuron edQTL in CCDC88A with educational attainment and a progenitor apaQTL in EP300 with schizophrenia, suggesting that genetically mediated post-transcriptional regulation during brain development leads to differences in brain function.

A-to-I RNA Editing and Hematopoiesis.

Adenosine to inosine (A-to-I) RNA editing plays essential roles in modulating normal development and homeostasis. This process is catalyzed by Adenosine Deaminase Acting on RNA (ADAR) family proteins. The most well-understood biological processes modulated by A-to-I editing are innate immunity and neurological development, attributed to ADAR1 and ADAR2 respectively. A-to-I editing by ADAR1 is also critical in regulating hematopoiesis. This review will focus on the role of A-to-I RNA editing and ADAR enzymes, particularly ADAR1, during normal hematopoiesis in humans and mice. Furthermore, we will discuss Adar1 mouse models that have been developed to understand the contribution of ADAR1 to hematopoiesis and its role in innate immune pathways.

Complete mitochondrial genome assembly of Zizania latifolia and comparative genome analysis.

Zizania latifolia (Griseb.) Turcz. ex Stapf has been cultivated as a popular aquatic vegetable in China due to its important nutritional, medicinal, ecological, and economic values. The complete mitochondrial genome (mitogenome) of Z. latifolia has not been previously studied and reported, which has hindered its molecular systematics and understanding of evolutionary processes. Here, we assembled the complete mitogenome of Z. latifolia and performed a comprehensive analysis including genome organization, repetitive sequences, RNA editing event, intercellular gene transfer, phylogenetic analysis, and comparative mitogenome analysis. The mitogenome of Z. latifolia was estimated to have a circular molecule of 392,219 bp and 58 genes consisting of three rRNA genes, 20 tRNA genes, and 35 protein-coding genes (PCGs). There were 46 and 20 simple sequence repeats (SSRs) with different motifs identified from the mitogenome and chloroplast genome of Z. latifolia, respectively. Furthermore, 49 homologous fragments were observed to transfer from the chloroplast genome to the mitogenome of Z. latifolia, accounting for 47,500 bp, presenting 12.1% of the whole mitogenome. In addition, there were 11 gene-containing homologous regions between the mitogenome and chloroplast genome of Z. latifolia. Also, approximately 85% of fragments from the mitogenome were duplicated in the Z. latifolia nuclear genome. Selection pressure analysis revealed that most of the mitochondrial genes were highly conserved except for ccmFc, ccmFn, matR, rps1, and rps3. A total of 93 RNA editing sites were found in the PCGs of the mitogenome. Z. latifolia and Oryza minuta are the most closely related, as shown by collinear analysis and the phylogenetic analysis. We found that repeat sequences and foreign sequences in the mitogenomes of Oryzoideae plants were associated with genome rearrangements. In general, the availability of the Z. latifolia mitogenome will contribute valuable information to our understanding of the molecular and genomic aspects of Zizania.

Comparative analysis of the mitochondrial genomes of four Dendrobium species (Orchidaceae) reveals heterogeneity in structure, synteny, intercellular gene transfer, and RNA editing.

The genus Dendrobium, part of the Orchidaceae family, encompasses species of significant medicinal, nutritional, and economic value. However, many Dendrobium species are threatened by environmental stresses, low seed germination rates, and overharvesting. Mitochondria generate the energy necessary for various plant life activities. Despite their importance, research on the mitochondrial genomes of Dendrobium species is currently limited. To address this gap, we performed a comprehensive genetic analysis of four Dendrobium species-D. flexicaule, D. nobile, D. officinale, and D. huoshanense-focusing on their mitochondrial and chloroplast genomes to elucidate their genetic architecture and support conservation efforts. We utilized advanced sequencing technologies, including Illumina for high-throughput sequencing and Nanopore for long-read sequencing capabilities. Our findings revealed the multichromosomal mitochondrial genome structures, with total lengths ranging from 596,506 bp to 772,523 bp. The mitochondrial genomes contained 265 functional genes, including 64-69 protein-coding genes, 23-28 tRNA genes, and 3 rRNA genes. We identified 647 simple sequence repeats (SSRs) and 352 tandem repeats, along with 440 instances of plastid-to-mitochondrial gene transfer. Additionally, we predicted 2,023 RNA editing sites within the mitochondrial protein-coding genes, predominantly characterized by cytosine-to-thymine transitions. Comparative analysis of mitochondrial DNA across the species highlighted 25 conserved genes, with evidence of positive selection in five genes: ccmFC, matR, mttB, rps2, and rps10. Phylogenetic assessments suggested a close sister relationship between D. nobile and D. huoshanense, and a similar proximity between D. officinale and D. flexicaule. This comprehensive genomic study provides a critical foundation for further exploration into the genetic mechanisms and biodiversity of Dendrobium species, contributing valuable insights for their conservation and sustainable utilization.

F11R RNA trinucleotide over-edited by ADAR in gastric and colorectal cancers: Cross-cohort validation, gene expression regulation, and diagnostic significance.

The F11 receptor (F11R) gene encoding junctional adhesion molecule A has been associated with gastric cancer (GC) and colorectal cancer (CRC), in which its role and regulation remain to be further elucidated. Recently F11R was also identified as a potential target of adenosine-to-inosine (A-to-I) mediated by the adenosine deaminases acting on RNA (ADARs). Herein, using RNA-Seq and experimental validation, our current study revealed an F11R RNA trinucleotide over-edited by ADAR, with its regulation of gene expression and clinical significance in four GC and three CRC cohorts. Our results found an over-edited AAA trinucleotide in an AluSg located in the F11R 3'-untranslated region (3'-UTR), which showed editing levels correlated with elevated ADAR expression across all GC and CRC cohorts in our study. Overexpression and knockdown of ADAR in GC and CRC cells, followed by RNA-Seq and Sanger sequencing, confirmed the ADAR-mediated F11R 3'-UTR trinucleotide editing, which potentially disrupted an RBM45 binding site identified by crosslinking immunoprecipitation sequencing (CLIP-seq) and regulated F11R expression in luciferase reporter assays. Moreover, the F11R trinucleotide editing showed promising predictive performance for diagnosing GC and CRC across GC and CRC cohorts. Our findings thus highlight both the potential biological and clinical significance of an ADAR-edited F11R trinucleotide in GC and CRC, providing new insights into its application as a novel diagnostic biomarker for both cancers.

The HPE1 RNA-binding protein modulates chloroplast RNA editing to promote photosynthesis under cold stress in Arabidopsis.

Cold stress has severe negative consequences for plant growth and crop yield. Here, we report that an Arabidopsis thaliana mutant that lacks the HPE1 gene, which encodes an RNA-binding protein, maintains higher photosynthetic activity under cold stress, together with higher accumulation of thylakoid proteins. We showed that HPE1 interacts with MORF2 and MORF9 and thereby mediates RNA editing in chloroplasts. Loss of HPE1 function increased the editing efficiency at four RNA editing sites, rpoC-488, ndhB-149, ndhB-746 and matK-706, under cold stress and altered the expression of nuclear photosynthesis-related genes and cold-responsive genes. We propose that HPE1-mediated RNA editing acts as a trigger for retrograde signaling that affects photosynthesis under cold stress.