Unveiling the A-to-I mRNA editing machinery and its regulation and evolution in fungi.

A-to-I mRNA editing in animals is mediated by ADARs, but the mechanism underlying sexual stage-specific A-to-I mRNA editing in fungi remains unknown. Here, we show that the eukaryotic tRNA-specific heterodimeric deaminase FgTad2-FgTad3 is responsible for A-to-I mRNA editing in Fusarium graminearum. This editing capacity relies on the interaction between FgTad3 and a sexual stage-specific protein called Ame1. Although Ame1 orthologs are widely distributed in fungi, the interaction originates in Sordariomycetes. We have identified key residues responsible for the FgTad3-Ame1 interaction. The expression and activity of FgTad2-FgTad3 are regulated through alternative promoters, alternative translation initiation, and post-translational modifications. Our study demonstrates that the FgTad2-FgTad3-Ame1 complex can efficiently edit mRNA in yeasts, bacteria, and human cells, with important implications for the development of base editors in therapy and agriculture. Overall, this study uncovers mechanisms, regulation, and evolution of RNA editing in fungi, highlighting the role of protein-protein interactions in modulating deaminase function.

Transfer learning enables identification of multiple types of RNA modifications using nanopore direct RNA sequencing.

Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.

Temporal landscape and translational regulation of A-to-I RNA editing in mouse retina development.

BACKGROUND: The significance of A-to-I RNA editing in nervous system development is widely recognized; however, its influence on retina development remains to be thoroughly understood. RESULTS: In this study, we performed RNA sequencing and ribosome profiling experiments on developing mouse retinas to characterize the temporal landscape of A-to-I editing. Our findings revealed temporal changes in A-to-I editing, with distinct editing patterns observed across different developmental stages. Further analysis showed the interplay between A-to-I editing and alternative splicing, with A-to-I editing influencing splicing efficiency and the quantity of splicing events. A-to-I editing held the potential to enhance translation diversity, but this came at the expense of reduced translational efficiency. When coupled with splicing, it could produce a coordinated effect on gene translation. CONCLUSIONS: Overall, this study presents a temporally resolved atlas of A-to-I editing, connecting its changes with the impact on alternative splicing and gene translation in retina development.

Ocular A-to-I RNA editing signatures associated with SARS-CoV-2 infection.

Ophthalmic manifestations have recently been observed in acute and post-acute complications of COVID-19 caused by SARS-CoV-2 infection. Our precious study has shown that host RNA editing is linked to RNA viral infection, yet ocular adenosine to inosine (A-to-I) RNA editing during SARS-CoV-2 infection remains uninvestigated in COVID-19. Herein we used an epitranscriptomic pipeline to analyze 37 samples and investigate A-to-I editing associated with SARS-CoV-2 infection, in five ocular tissue types including the conjunctiva, limbus, cornea, sclera, and retinal organoids. Our results revealed dramatically altered A-to-I RNA editing across the five ocular tissues. Notably, the transcriptome-wide average level of RNA editing was increased in the cornea but generally decreased in the other four ocular tissues. Functional enrichment analysis showed that differential RNA editing (DRE) was mainly in genes related to ubiquitin-dependent protein catabolic process, transcriptional regulation, and RNA splicing. In addition to tissue-specific RNA editing found in each tissue, common RNA editing was observed across different tissues, especially in the innate antiviral immune gene MAVS and the E3 ubiquitin-protein ligase MDM2. Analysis in retinal organoids further revealed highly dynamic RNA editing alterations over time during SARS-CoV-2 infection. Our study thus suggested the potential role played by RNA editing in ophthalmic manifestations of COVID-19, and highlighted its potential transcriptome impact, especially on innate immunity.

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.

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.

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.

Decoding the language of immunity.

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

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.

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.

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.

Changes in ADAR RNA editing patterns in CMV and ZIKV congenital infections.

BACKGROUND: RNA editing is a process that increases transcriptome diversity, often through Adenosine Deaminases Acting on RNA (ADARs) that catalyze the deamination of adenosine to inosine. ADAR editing plays an important role in regulating brain function and immune activation, and is dynamically regulated during brain development. Additionally, the ADAR1 p150 isoform is induced by interferons in viral infection and plays a role in antiviral immune response. However, the question of how virus-induced ADAR expression affects host transcriptome editing remains largely unanswered. This question is particularly relevant in the context of congenital infections, given the dynamic regulation of ADAR editing during brain development, the importance of this editing for brain function, and subsequent neurological symptoms of such infections, including microcephaly, sensory issues, and other neurodevelopmental abnormalities. Here, we begin to address this question, examining ADAR expression in publicly available datasets of congenital infections of human cytomegalovirus (HCMV) microarray expression data, as well as mouse cytomegalovirus (MCMV) and mouse/ human induced pluripotent neuroprogenitor stem cell (hiNPC) Zika virus (ZIKV) RNA-seq data. RESULTS: We found that in all three datasets, ADAR1 was overexpressed in infected samples compared to uninfected samples. In the RNA-seq datasets, editing rates were also analyzed. In all mouse infections cases, the number of editing sites was significantly increased in infected samples, albeit this was not the case for hiNPC ZIKV samples. Mouse ZIKV samples showed altered editing of well-established protein-recoding sites such as Gria3, Grik5, and Nova1, as well as editing sites that may impact miRNA binding. CONCLUSIONS: Our findings provide evidence for changes in ADAR expression and subsequent dysregulation of ADAR editing of host transcriptomes in congenital infections. These changes in editing patterns of key neural genes have potential significance in the development of neurological symptoms, thus contributing to neurodevelopmental abnormalities. Further experiments should be performed to explore the full range of editing changes that occur in different congenital infections, and to confirm the specific functional consequences of these editing changes.

The Many Roles of A-to-I RNA Editing in Animals: Functional or Adaptive?

Metazoan adenosine-to-inosine (A-to-I) RNA editing is a highly conserved mechanism that diversifies the transcriptome by post-transcriptionally converting adenosine to inosine. Millions of editing sites have been identified in different species and, based on abnormal editing observed in various disorders, it is intuitive to conclude that RNA editing is both functional and adaptive. In this review, we propose the following major points: (1) "Function/functional" only represents a molecular/phenotypic consequence and is not necessarily connected to "adaptation/adaptive"; (2) Adaptive editing should be judged in the light of evolution and emphasize advantages of temporal-spatial flexibility; (3) Adaptive editing could, in theory, be extended from nonsynonymous sites to all potentially functional sites. This review seeks to conceptually bridge the gap between molecular biology and evolutionary biology and provide a more objective understanding on the biological functions and evolutionary significance of RNA editing.

Genome-Wide Analysis on Driver and Passenger RNA Editing Sites Suggests an Underestimation of Adaptive Signals in Insects.

Adenosine-to-inosine (A-to-I) RNA editing leads to a similar effect to A-to-G mutations. RNA editing provides a temporo-spatial flexibility for organisms. Nonsynonymous (Nonsyn) RNA editing in insects is over-represented compared with synonymous (Syn) editing, suggesting adaptive signals of positive selection on Nonsyn editing during evolution. We utilized the brain RNA editome of Drosophila melanogaster to systematically study the LD (r2) between editing sites and infer its impact on the adaptive signals of RNA editing. Pairs of editing sites (PESs) were identified from the transcriptome. For CDS PESs of two consecutive editing sites, their occurrence was significantly biased to type-3 PES (Syn-Nonsyn). The haplotype frequency of type-3 PES exhibited a significantly higher abundance of AG than GA, indicating that the rear Nonsyn site is the driver that promotes the editing of the front Syn site (passenger). The exclusion of passenger Syn sites dramatically amplifies the adaptive signal of Nonsyn RNA editing. Our study for the first time quantitatively demonstrates that the linkage between RNA editing events comes from hitchhiking effects and leads to the underestimation of adaptive signals for Nonsyn editing. Our work provides novel insights for studying the evolutionary significance of RNA editing events.

Adenosine Deaminase Acting on RNA (ADAR) Enzymes: A Journey from Weird to Wondrous.

ConspectusThe adenosine deaminase acting on RNA (ADAR) enzymes that catalyze the conversion of adenosine to inosine in double-stranded (ds)RNA are evolutionarily conserved and are essential for many biological functions including nervous system function, hematopoiesis, and innate immunity. Initially it was assumed that the wide-ranging biological roles of ADARs are due to inosine in mRNA being read as guanosine by the translational machinery, allowing incomplete RNA editing in a target codon to generate two different proteins from the same primary transcript. In humans, there are approximately seventy-six positions that undergo site-specific editing in tissues at greater than 20% efficiency that result in recoding. Many of these transcripts are expressed in the central nervous system (CNS) and edited by ADAR2. Exploiting mouse genetic models revealed that transgenic mice lacking the gene encoding Adar2 die within 3 weeks of birth. Therefore, the role of ADAR2 in generating protein diversity in the nervous system is clear, but why is ADAR RNA editing activity essential in other biological processes, particularly editing mainly involving ADAR1? ADAR1 edits human transcripts having embedded Alu element inverted repeats (AluIRs), but the link from this activity to innate immunity activation was elusive. Mice lacking the gene encoding Adar1 are embryonically lethal, and a major breakthrough was the discovery that the role of Adar1 in innate immunity is due to its ability to edit such repetitive element inverted repeats which have the ability to form dsRNA in transcripts. The presence of inosine prevents activation of the dsRNA sensor melanoma differentiation-associated protein 5 (Mda5). Thus, inosine helps the cell discriminate self from non-self RNA, acting like a barcode on mRNA. As innate immunity is key to many different biological processes, the basis for this widespread biological role of the ADAR1 enzyme became evident.Our group has been studying ADARs from the outset of research on these enzymes. In this Account, we give a historical perspective, moving from the initial purification of ADAR1 and ADAR2 and cloning of their encoding genes up to the current research focus in the field and what questions still remain to be addressed. We discuss the characterizations of the proteins, their localizations, posttranslational modifications, and dimerization, and how all of these affect their biological activities. Another aspect we explore is the use of mouse and Drosophila genetic models to study ADAR functions and how these were crucial in determining the biological functions of the ADAR proteins. Finally, we describe the severe consequences of rare mutations found in the human genes encoding ADAR1 and ADAR2.

Incorporation of inosine into DNA by human polymerase eta (Polη): kinetics of nucleotide misincorporation and structural basis for the mutagenicity.

Inosine, a purine nucleoside containing the hypoxanthine (HX) nucleobase, can form in DNA via hydrolytic deamination of adenine. Due to its structural similarity to guanine and the geometry of Watson-Crick base pairs, inosine can mispair with cytosine upon catalysis by DNA polymerases, leading to AT → GC mutations. Additionally, inosine plays an essential role in purine nucleotide biosynthesis, and inosine triphosphate is present in living cells. In a recent publication, Averill and Jung examined the possibility of polη catalyzed incorporation of deoxyinosine triphosphate (dITP) across dC and dT in a DNA template. They found that dITP can be incorporated across C or T, with the ratio of 13.7. X ray crystallography studies revealed that the mutagenic incorporation of dITP by human polη was affected by several factors including base pair geometry in the active site of the polymerase, tautomerization of nucleobases, and the interaction of the incoming dITP nucleotide with active site residues of polη. This study demonstrates that TLS incorporation of inosine monophosphate (IMP) into growing DNA chains contributes to its mutagenic potential in cells.

yaaJ, the tRNA-Specific Adenosine Deaminase, Is Dispensable in Bacillus subtilis.

Post-transcriptional modifications of tRNA are crucial for their core function. The inosine (I; 6-deaminated adenosine) at the first position in the anticodon of tRNAArg(ICG) modulates the decoding capability and is generally considered essential for reading CGU, CGC, and CGA codons in eubacteria. We report here that the Bacillus subtilis yaaJ gene encodes tRNA-specific adenosine deaminase and is non-essential for viability. A ß-galactosidase reporter assay revealed that the translational activity of CGN codons was not impaired in the yaaJ-deletion mutant. Furthermore, tRNAArg(CCG) responsible for decoding the CGG codon was dispensable, even in the presence or absence of yaaJ. These results strongly suggest that tRNAArg with either the anticodon ICG or ACG has an intrinsic ability to recognize all four CGN codons, providing a fundamental concept of non-canonical wobbling mediated by adenosine and inosine nucleotides in the anticodon. This is the first example of the four-way wobbling by inosine nucleotide in bacterial cells. On the other hand, the absence of inosine modification induced +1 frameshifting, especially at the CGA codon. Additionally, the yaaJ deletion affected growth and competency. Therefore, the inosine modification is beneficial for translational fidelity and proper growth-phase control, and that is why yaaJ has been actually conserved in B. subtilis.

Autorecoding A-to-I RNA editing sites in the Adar gene underwent compensatory gains and losses in major insect clades.

As one of the most prevalent RNA modifications in animals, adenosine-to-inosine (A-to-I) RNA editing facilitates the environmental adaptation of organisms by diversifying the proteome in a temporal-spatial manner. In flies and bees, the editing enzyme Adar has independently gained two different autorecoding sites that form an autofeedback loop, stabilizing the overall editing efficiency. This ensures cellular homeostasis by keeping the normal function of target genes. However, in a broader range of insects, the evolutionary dynamics and significance of this Adar autoregulatory mechanism are unclear. We retrieved the genomes of 377 arthropod species covering the five major insect orders (Hemiptera, Hymenoptera, Coleoptera, Diptera, and Lepidoptera) and aligned the Adar autorecoding sites across all genomes. We found that the two autorecoding sites underwent compensatory gains and losses during the evolution of two orders with the most sequenced species (Diptera and Hymenoptera), and that the two editing sites were mutually exclusive among them: One editable site is significantly linked to another uneditable site. This autorecoding mechanism of Adar could flexibly diversify the proteome and stabilize global editing activity. Many insects independently selected different autorecoding sites to achieve a feedback loop and regulate the global RNA editome, revealing an interesting phenomenon during evolution. Our study reveals the evolutionary force acting on accurate regulation of RNA editing activity in insects and thus deepens our understanding of the functional importance of RNA editing in environmental adaptation and evolution.