Dysregulated RNA editing of EIF2AK2 in polycystic ovary syndrome: clinical relevance and functional implications.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive ages. Our previous study has implicated a possible link between RNA editing and PCOS, yet the actual role of RNA editing, its association with clinical features, and the underlying mechanisms remain unclear. METHODS: Ten RNA-Seq datasets containing 269 samples of multiple tissue types, including granulosa cells, T helper cells, placenta, oocyte, endometrial stromal cells, endometrium, and adipose tissues, were retrieved from public databases. Peripheral blood samples were collected from twelve PCOS and ten controls and subjected to RNA-Seq. Transcriptome-wide RNA-Seq data analysis was conducted to identify differential RNA editing (DRE) between PCOS and controls. The functional significance of DRE was evaluated by luciferase reporter assays and overexpression in human HEK293T cells. Dehydroepiandrosterone and lipopolysaccharide were used to stimulate human KGN granulosa cells to evaluate gene expression. RESULTS: RNA editing dysregulations across multiple tissues were found to be associated with PCOS in public datasets. Peripheral blood transcriptome analysis revealed 798 DRE events associated with PCOS. Through weighted gene co-expression network analysis, our results revealed a set of hub DRE events in PCOS blood. A DRE event in the eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2:chr2:37,100,559) was associated with PCOS clinical features such as luteinizing hormone (LH) and the ratio of LH over follicle-stimulating hormone. Luciferase assays, overexpression, and knockout of RNA editing enzyme adenosine deaminase RNA specific (ADAR) showed that the ADAR-mediated editing cis-regulated EIF2AK2 expression. EIAF2AK2 showed a higher expression after dehydroepiandrosterone and lipopolysaccharide stimulation, triggering changes in the downstrean MAPK pathway. CONCLUSIONS: Our study presented the first evidence of cross-tissue RNA editing dysregulation in PCOS and its clinical associations. The dysregulation of RNA editing mediated by ADAR and the disrupted target EIF2AK2 may contribute to PCOS development via the MPAK pathway, underlining such epigenetic mechanisms in the disease.

Insights into structure, codon usage, repeats, and RNA editing of the complete mitochondrial genome of Perilla frutescens (Lamiaceae).

Perilla frutescens (L.) Britton, a member of the Lamiaceae family, stands out as a versatile plant highly valued for its unique aroma and medicinal properties. Additionally, P. frutescens seeds are rich in Îs-linolenic acid, holding substantial economic importance. While the nuclear and chloroplast genomes of P. frutescens have already been documented, the complete mitochondrial genome sequence remains unreported. To this end, the sequencing, annotation, and assembly of the entire Mitochondrial genome of P. frutescens were hereby conducted using a combination of Illumina and PacBio data. The assembled P. frutescens mitochondrial genome spanned 299,551 bp and exhibited a typical circular structure, involving a GC content of 45.23%. Within the genome, a total of 59 unique genes were identified, encompassing 37 protein-coding genes, 20 tRNA genes, and 2 rRNA genes. Additionally, 18 introns were observed in 8 protein-coding genes. Notably, the codons of the P. frutescens mitochondrial genome displayed a notable A/T bias. The analysis also revealed 293 dispersed repeat sequences, 77 simple sequence repeats (SSRs), and 6 tandem repeat sequences. Moreover, RNA editing sites preferentially produced leucine at amino acid editing sites. Furthermore, 70 sequence fragments (12,680 bp) having been transferred from the chloroplast to the mitochondrial genome were identified, accounting for 4.23% of the entire mitochondrial genome. Phylogenetic analysis indicated that among Lamiaceae plants, P. frutescens is most closely related to Salvia miltiorrhiza and Platostoma chinense. Meanwhile, inter-species Ka/Ks results suggested that Ka/Ks < 1 for 28 PCGs, indicating that these genes were evolving under purifying selection. Overall, this study enriches the mitochondrial genome data for P. frutescens and forges a theoretical foundation for future molecular breeding research.

Molecular mechanisms of non-genetic aberrant peptide production in cancer.

The cancer peptidome has long been known to be altered by genetic mutations. However, more recently, non-genetic polypeptide mutations have also been related to cancer cells. These non-genetic mutations occur post-t30ranscriptionally, leading to the modification of the peptide primary structure, while the corresponding genes remain unchanged. Three main processes participate in the production of these aberrant proteins: mRNA alternative splicing, mRNA editing, and mRNA aberrant translation. In this review, we summarize the molecular mechanisms underlying these processes and the recent findings on the functions of the aberrant proteins, as well as their exploitability as new therapeutic targets due to their specific enrichment in cancer cells. These non-genetic aberrant polypeptides represent a source of novel cancer cell targets independent from their level of mutational burden, still to be exhaustively explored.

Development and assessment of an RNA editing-based risk model for the prognosis of cervical cancer patients.

RNA editing, as an epigenetic mechanism, exhibits a strong correlation with the occurrence and development of cancers. Nevertheless, few studies have been conducted to investigate the impact of RNA editing on cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). In order to study the connection between RNA editing and CESC patients' prognoses, we obtained CESC-related information from The Cancer Genome Atlas (TCGA) database and randomly allocated the patients into the training group or testing group. An RNA editing-based risk model for CESC patients was established by Cox regression analysis and least absolute shrinkage and selection operator (LASSO). According to the median score generated by this RNA editing-based risk model, patients were categorized into subgroups with high and low risks. We further constructed the nomogram by risk scores and clinical characteristics and analyzed the impact of RNA editing levels on host gene expression levels and adenosine deaminase acting on RNA. Finally, we also compared the biological functions and pathways of differentially expressed genes (DEGs) between different subgroups by enrichment analysis. In this risk model, we screened out 6 RNA editing sites with significant prognostic value. The constructed nomogram performed well in forecasting patients' prognoses. Furthermore, the level of RNA editing at the prognostic site exhibited a strong correlation with host gene expression. In the high-risk subgroup, we observed multiple biological functions and pathways associated with immune response, cell proliferation, and tumor progression. This study establishes an RNA editing-based risk model that helps forecast patients' prognoses and offers a new understanding of the underlying mechanism of RNA editing in CESC.

Insights into U-to-C RNA editing from the lycophyte Phylloglossum drummondii.

The lycophyte Phylloglossum drummondii is the sole inhabitant of its genus in the Huperzioideae group and one of a small minority of plants which perform uridine to cytidine RNA editing. We assembled the P. drummondii chloroplast and mitochondrial genomes and used RNA sequence data to build a comprehensive profile of organellar RNA editing events. In addition to many C-to-U editing events in both organelles, we found just four U-to-C editing events in the mitochondrial transcripts cob, nad1, nad5 and rpl2. These events are conserved in related lycophytes in the genera Huperzia and Phlegmariurus. De novo transcriptomes for three of these lycophytes were assembled to search for putative U-to-C RNA editing enzymes. Four putative U-to-C editing factors could be matched to the four mitochondrial U-to-C editing sites. Due to the unusually few numbers of U-to-C RNA editing sites, P. drummondii and related lycophytes are useful models for studying this poorly understood mechanism.

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.

Is RNA editing truly absent in the complex thalloid liverworts (Marchantiopsida)? Evidence of extensive RNA editing from Cyathodium cavernarum.

RNA editing is a crucial modification in plants' organellar transcripts that converts cytidine to uridine (C-to-U; and sometimes uridine to cytidine) in RNA molecules. This post-transcriptional process is controlled by the PLS-class protein with a DYW domain, which belongs to the pentatricopeptide repeat (PPR) protein family. RNA editing is widespread in land plants; however, complex thalloid liverworts (Marchantiopsida) are the only group reported to lack both RNA editing and DYW-PPR protein. The liverwort Cyathodium cavernarum (Marchantiopsida, Cyathodiaceae), typically found in cave habitats, was newly found to have 129 C-to-U RNA editing sites in its chloroplast and 172 sites in its mitochondria. The Cyathodium genus, specifically C. cavernarum, has a large number of PPR editing factor genes, including 251 DYW-type PPR proteins. These DYW-type PPR proteins may be responsible for C-to-U RNA editing in C. cavernarum. Cyathodium cavernarum possesses both PPR DYW proteins and RNA editing. Our analysis suggests that the remarkable RNA editing capability of C. cavernarum may have been acquired alongside the emergence of DYW-type PPR editing factors. These findings provide insight into the evolutionary pattern of RNA editing in land plants.

Sexual stage-specific A-to-I mRNA editing is mediated by tRNA-editing enzymes in fungi.

A-to-I RNA editing catalyzed by adenosine-deaminase-acting-on-RNA (ADARs) was assumed to be unique to metazoans because fungi and plants lack ADAR homologs. However, genome-wide messenger RNA (mRNA) editing was found to occur specifically during sexual reproduction in filamentous ascomycetes. Because systematic characterization of adenosine/cytosine deaminase genes has implicated the involvement of TAD2 and TAD3 orthologs in A-to-I editing, in this study, we used genetic and biochemical approaches to characterize the role of FgTAD2, an essential adenosine-deaminase-acting-on-tRNA (ADAT) gene, in mRNA editing in Fusarium graminearum. FgTAD2 had a sexual-stage-specific isoform and formed heterodimers with enzymatically inactive FgTAD3. Using a repeat-induced point (RIP) mutation approach, we identified 17 mutations in FgTAD2 that affected mRNA editing during sexual reproduction but had no effect on transfer RNA (tRNA) editing and vegetative growth. The functional importance of the H352Y and Q375*(nonsense) mutations in sexual reproduction and mRNA editing were confirmed by introducing specific point mutations into the endogenous FgTAD2 allele in the wild type. An in vitro assay was developed to show that FgTad2-His proteins purified from perithecia, but not from vegetative hyphae, had mRNA editing activities. Moreover, the H352Y mutation affected the enzymatic activity of FgTad2 to edit mRNA but had no effect on its ADAT activity. We also identified proteins co-purified with FgTad2-His by mass spectrometry analysis and found that two of them have the RNA recognition motif. Taken together, genetic and biochemical data from this study demonstrated that FgTad2, an ADAT, catalyzes A-to-I mRNA editing with the stage-specific isoform and cofactors during sexual reproduction in fungi.

Programmable RNA N6-methyladenosine editing with CRISPR/dCas13a in plants.

N6-methyladenonsine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual m6A modification to reveal its function and the phenotypic effects is still lacking in plants. Here, we describe the construction and characterization of programmable m6A editing tools by fusing the m6A writers, the core catalytic domain of the MTA and MTB complex, and the AlkB homologue 5 (ALKBH5) eraser, to catalytically dead Cas13a (dCas13a) to edit individual m6A sites on mRNAs. We demonstrated that our m6A editors could efficiently and specifically deposit and remove m6A modifications on specific RNA transcripts in both Nicotiana benthamiana and Arabidopsis thaliana. Moreover, we found that targeting SHORT-ROOT (SHR) transcripts with a methylation editor could significantly increase its m6A levels with limited off-target effects and promote its degradation. This leads to a boost in plant growth with enlarged leaves and roots, increased plant height, plant biomass, and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study the functions of individual m6A modifications in plants, and may also have potential applications for future crop improvement.

MORF9-dependent specific plastid RNA editing inhibits root growth under sugar starvation in Arabidopsis.

Multiple organellar RNA editing factor (MORF) complex was shown to be highly associated with C-to-U RNA editing of vascular plant editosome. However, mechanisms by which MORF9-dependent plastid RNA editing controls plant development and responses to environmental alteration remain obscure. In this study, we found that loss of MORF9 function impaired PSII efficiency, NDH activity, and carbohydrate production, rapidly promoted nuclear gene expression including sucrose transporter and sugar/energy responsive genes, and attenuated root growth under sugar starvation conditions. Sugar repletion increased MORF9 and MORF2 expression in wild-type seedlings and reduced RNA editing of matK-706, accD-794, ndhD-383 and ndhF-290 in the morf9 mutant. RNA editing efficiency of ndhD-383 and ndhF-290 sites was diminished in the gin2/morf9 double mutants, and that of matK-706, accD-794, ndhD-383 and ndhF-290 sites were significantly diminished in the snrk1/morf9 double mutants. In contrast, overexpressing HXK1 or SnRK1 promoted RNA editing rate of matK-706, accD-794, ndhD-383 and ndhF-290 in leaves of morf9 mutants, suggesting that HXK1 partially impacts MORF9 mediated ndhD-383 and ndhF-290 editing, while SnRK1 may only affect MORF9-mediated ndhF-290 site editing. Collectively, these findings suggest that sugar and/or its intermediary metabolites impair MORF9-dependent plastid RNA editing resulting in derangements of plant root development.

Programmable RNA base editing with photoactivatable CRISPR-Cas13.

CRISPR-Cas13 is widely used for programmable RNA interference, imaging, and editing. In this study, we develop a light-inducible Cas13 system called paCas13 by fusing Magnet with fragment pairs. The most effective split site, N351/C350, was identified and found to exhibit a low background and high inducibility. We observed significant light-induced perturbation of endogenous transcripts by paCas13. We further present a light-inducible base-editing system, herein called the padCas13 editor, by fusing ADAR2 to catalytically inactive paCas13 fragments. The padCas13 editor enabled reversible RNA editing under light and was effective in editing A-to-I and C-to-U RNA bases, targeting disease-relevant transcripts, and fine-tuning endogenous transcripts in mammalian cells in vitro. The padCas13 editor was also used to adjust post-translational modifications and demonstrated the ability to activate target transcripts in a mouse model in vivo. We therefore present a light-inducible RNA-modulating technique based on CRISPR-Cas13 that enables target RNAs to be diversely manipulated in vitro and in vivo, including through RNA degradation and base editing. The approach using the paCas13 system can be broadly applicable to manipulating RNA in various disease states and physiological processes, offering potential additional avenues for research and therapeutic development.

Characterization and analysis of multi-organ full-length transcriptomes in Sphaeropteris brunoniana and Alsophila latebrosa highlight secondary metabolism and chloroplast RNA editing pattern of tree ferns.

BACKGROUND: Sphaeropteris brunoniana and Alsophila latebrosa are both old relict and rare tree ferns, which have experienced the constant changes of climate and environment. However, little is known about their high-quality genetic information and related research on environmental adaptation mechanisms of them. In this study, combined with PacBio and Illumina platforms, transcriptomic analysis was conducted on the roots, rachis, and pinna of S. brunoniana and A. latebrosa to identify genes and pathways involved in environmental adaptation. Additionally, based on the transcriptomic data of tree ferns, chloroplast genes were mined to analyze their gene expression levels and RNA editing events. RESULTS: In the study, we obtained 11,625, 14,391 and 10,099 unigenes of S. brunoniana root, rachis, and pinna, respectively. Similarly, a total of 13,028, 11,431 and 12,144 unigenes were obtained of A. latebrosa root, rachis, and pinna, respectively. According to the enrichment results of differentially expressed genes, a large number of differentially expressed genes were enriched in photosynthesis and secondary metabolic pathways of S. brunoniana and A. latebrosa. Based on gene annotation results and phenylpropanoid synthesis pathways, two lignin synthesis pathways (H-lignin and G-lignin) were characterized of S. brunoniana. Among secondary metabolic pathways of A. latebrosa, three types of WRKY transcription factors were identified. Additionally, based on transcriptome data obtained in this study, reported transcriptome data, and laboratory available transcriptome data, positive selection sites were identified from 18 chloroplast protein-coding genes of four tree ferns. Among them, RNA editing was found in positive selection sites of four tree ferns. RNA editing affected the protein secondary structure of the rbcL gene. Furthermore, the expression level of chloroplast genes indicated high expression of genes related to the chloroplast photosynthetic system in all four species. CONCLUSIONS: Overall, this work provides a comprehensive transcriptome resource of S. brunoniana and A. latebrosa, laying the foundation for future tree fern research.

Adaptive advantages of restorative RNA editing in fungi for resolving survival-reproduction trade-offs.

RNA editing in various organisms commonly restores RNA sequences to their ancestral state, but its adaptive advantages are debated. In fungi, restorative editing corrects premature stop codons in pseudogenes specifically during sexual reproduction. We characterized 71 pseudogenes and their restorative editing in Fusarium graminearum, demonstrating that restorative editing of 16 pseudogenes is crucial for germ tissue development in fruiting bodies. Our results also revealed that the emergence of premature stop codons is facilitated by restorative editing and that premature stop codons corrected by restorative editing are selectively favored over ancestral amino acid codons. Furthermore, we found that ancestral versions of pseudogenes have antagonistic effects on reproduction and survival. Restorative editing eliminates the survival costs of reproduction caused by antagonistic pleiotropy and provides a selective advantage in fungi. Our findings highlight the importance of restorative editing in the evolution of fungal complex multicellularity and provide empirical evidence that restorative editing serves as an adaptive mechanism enabling the resolution of genetic trade-offs.

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.

ADAR-mediated RNA editing regulates PVR immune checkpoint in colorectal cancer.

Recent studies have revealed that tumor immunotherapy resistance is influenced by ADAR-mediated RNA editing, but its targets remain unelucidated. Our current study identified the poliovirus receptor (PVR) oncogene, which encodes an immune checkpoint in colorectal cancer (CRC), as a potential target for RNA editing. We performed transcriptome sequencing analysis and experimental validation in two Chinese CRC cohorts. PVR and ADAR expressions significantly increased in CRC tumors and showed positive correlations in both cohorts, coupled with upregulated PVR RNA editing in CRC tumors. Manipulation of ADAR expression by over-expression or knockdown substantially changed PVR expression and RNA editing in HTC116 CRC cells. Luciferase reporter and actinomycin D assays further revealed that RNA editing in PVR 3'-UTR could upregulate PVR RNA expression, probably by increasing the RNA stability. By increasing PVR expression, ADAR-mediate RNA editing might contribute to tumor- and immune-related gene functions and pathways in CRC. Moreover, a signature combining PVR RNA editing and expression showed promising predictive performance in CRC diagnosis in both Chinese CRC cohorts. Our findings thus highlight the importance of ADAR-mediated RNA editing in PVR up-regulation in CRC tumors and provide new insight into the application of PVR RNA editing as a novel diagnostic biomarker for CRC.

PlantC2U: deep learning of cross-species sequence landscapes predicts plastid C-to-U RNA editing in plants.

In plants, C-to-U RNA editing mainly occurs in plastid and mitochondrial transcripts, which contributes to a complex transcriptional regulatory network. More evidence reveals that RNA editing plays critical roles in plant growth and development. However, accurate detection of RNA editing sites using transcriptome sequencing data alone is still challenging. In the present study, we develop PlantC2U, which is a convolutional neural network, to predict plastid C-to-U RNA editing based on the genomic sequence. PlantC2U achieves >95% sensitivity and 99% specificity, which outperforms the PREPACT tool, random forests, and support vector machines. PlantC2U not only further checks RNA editing sites from transcriptome data to reduce possible false positives, but also assesses the effect of different mutations on C-to-U RNA editing based on the flanking sequences. Moreover, we found the patterns of tissue-specific RNA editing in the mangrove plant Kandelia obovata, and observed reduced C-to-U RNA editing rates in the cold stress response of K. obovata, suggesting their potential regulatory roles in plant stress adaptation. In addition, we present RNAeditDB, available online at https://jasonxu.shinyapps.io/RNAeditDB/. Together, PlantC2U and RNAeditDB will help researchers explore the RNA editing events in plants and thus will be of broad utility for the plant research community.