Editing Sites Research Articles

Comprehensive analysis of the mitochondrial genome of Ziziphus mauritiana Lam.: Investigating sequence structure, repeat-mediated recombination, chloroplast-derived transfer, RNA editing, and evolution

Ziziphus mauritiana Lam. is a valuable semi-deciduous tropical fruit tree with economic, medicinal, nutritional, and environmental significance. While the chloroplast genome of Z. mauritiana has been studied, the decoding of its mitochondrial genome (mtGenome) is crucial for various research areas, including molecular phylogeny and functional genomics. In this study, we extracted high-quality genomic DNA from the green leaves of Z. mauritiana and assembled the mtGenome by integrating sequencing data from both long and short reads. The Z. mauritiana mtGenome spans 324,124 bp with a 45.43 % GC content and exhibits six distinct conformations alongside the main circular sequence due to three pairs of repetitive fragments that facilitate recombination. RNA editing events resulted in changes in the physicochemical properties of amino acids, with genes expressed at higher levels showing an increased frequency of editing sites. Chloroplast-to-mitochondria horizontal gene transfers occurred in Z. mauritiana; however, these transferred fragments had few mutations, posing a challenge in determining the exact timing of these transfers. Phylogenetic analysis highlights a slower evolution rate of mitochondrial genes compared to chloroplast genes. These insights greatly enhance our understanding of the dynamics of the Z. mauritiana mtGenome, including recombination, gene expression, evolutionary processes, and more. The data and results of this study will serve as valuable mtGenome data resources within the Rhamnaceae family, thereby facilitating further genomics-based studies on Z. mauritiana.

Comprehensive assembly and comparative examination of the full mitochondrial genome in Castanea mollissima Blume

The Chinese chestnut, Castanea mollissima Blume, a nut-bearing tree native to China and North Korea, belongs to the Fagaceae family. As an important genetic resource, C. mollissima is vital in enhancing edible chestnut varieties and offers significant insights into the origin and evolution of chestnut species. While the chloroplast genome of C. mollissima has been sequenced, its mitochondrial genome (mitogenome) remains largely uncharted. In this study, we have characterized the C. mollissima mitogenome, assembling it utilizing reads from both BGI and Nanopore sequencing platforms, and conducted a comparative analysis with the mitochondrial genomes of closely related species. The mitogenome of C. mollissima manifests a polycyclic structure consisting of two circular molecules measuring 363,232 bp and 24,806 bp, respectively. This genome encompasses 35 unique protein-coding genes, 19 tRNA genes, and three rRNA genes. A total of 139 SSRs were identified throughout the entire C. mollissima mitogenome. Furthermore, the combined length of homologous fragments between the chloroplast and mitochondrial genomes was 5766 bp, constituting 1.49% of the mitogenome. We also predicted 484 RNA editing sites in C. mollissima, demonstrating C-to-U RNA editing. Phylogenetic analysis of related species' mitogenomes showed that C. mollissima was closely related to Lithocarpus litseifolius (Hance) Chun and Quercus acutissima Carruth. Interestingly, the mitogenome sequences of C. mollissima, L. litseifolius, Q. acutissima, Fagus sylvatica L., and Juglans mandshurica Maxim did not show conservation in their alignments, indicating frequent genome reorganization. This report marks the inaugural study of the C. mollissima mitogenome, serving as a benchmark genome for economically significant plants within the Castanea genus. Moreover, it supplies invaluable information that can guide future molecular breeding efforts and contribute to the broader understanding of chestnut genomics.

Detection and editing of the updated Arabidopsis plastid- and mitochondrial-encoded proteomes through PeptideAtlas.

Arabidopsis (Arabidopsis thaliana) ecotype Col-0 has plastid and mitochondrial genomes encoding over 100 proteins. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of RNA editing at the protein level is largely unexplored due to the complexities of detection. Here, we assembled the nonredundant set of identifiers, their correct protein sequences, and 452 predicted nonsynonymous editing sites of which 56 are edited at lower frequency. We then determined accumulation of edited and/or unedited proteoforms by searching ∼259 million raw tandem MS spectra from ProteomeXchange, which is part of PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). We identified all mitochondrial proteins and all except 3 plastid-encoded proteins (NdhG/Ndh6, PsbM, and Rps16), but no proteins predicted from the 4 ORFs were identified. We suggest that Rps16 and 3 of the ORFs are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical detection challenges. We detected 167 predicted edit sites at the proteome level. Minor frequency sites were edited at low frequency at the protein level except for cytochrome C biogenesis 382 at residue 124 (Ccb382-124). Major frequency sites (>50% editing of RNA) only accumulated in edited form (>98% to 100% edited) at the protein level, with the exception of Rpl5-22. We conclude that RNA editing for major editing sites is required for stable protein accumulation.

Multichromosomal mitochondrial genome of Punica granatum: comparative evolutionary analysis and gene transformation from chloroplast genomes

BackgroundPunica granatum is a fundamentally important fruit tree that has important economic, medicinal and ornamental properties. At present, there are few reports on the mitochondrial genome of pomegranate. Hence, in this study the P. granatum mitogenome was sequenced and assembled to further understanding of organization, variation, and evolution of mitogenomes of this tree species.ResultsThe genome structure was multi-chromosomes with seven circular contigs, measuring 382,774 bp in length with a 45.91% GC content. It contained 74 genes, including 46 protein-coding genes, 25 tRNA genes, and three rRNA genes. There were 188 pairs of dispersed repeats with lengths of 30 or greater, primarily consisting of reverse complementary repeats. The mitogenome analysis identified 114SSRs and 466 RNA editing sites. Analyses of codon usage, nucleotide diversity and gene migration from chloroplast to mitochondrial were also conducted. The collinear and comparative analysis of mitochondrial structures between P. granatum and its proximal species indicated that P. granatum ‘Taishanhong’ was closely related to P. granatum ‘Qingpitian’ and Lagerstroemia indica. Phylogenetic examination based on the mitogenome also confirmed the evolutionary relationship.ConclusionThe results offered crucial information on the evolutionary biology of pomegranate and highlighted ways to promote the utilization of the species’ germplasm.

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.

CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.

CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.

Unbranched rod-like RNA is required for RNA editing of hepatitis delta virus genotype 2 and genotype 4

RNA editing of the hepatitis delta virus (HDV) is essential for generating the large delta antigen, which is crucial for virion assembly. In HDV genotype 1 (HDV-1), editing occurs within the context of the unbranched rod-like structure characteristic of HDV RNA, while RNA editing in HDV-3 requires a branched double-hairpin structure. The regulation of RNA editing in HDV-2 and HDV-4 remains uncertain. Based on predictions of the unbranched rod-like RNA structures of HDV-2 and HDV-4, the editing site occurs as an A.C mismatch pair, surrounded by four base pairs upstream and two base pairs downstream of the editing site, respectively. To investigate HDV-2 and HDV-4 RNA editing, cultured cells were transfected with non-replicating editing reporters carrying wild-type sequences or specific mutations. The results revealed that the editing rates observed for wild-type HDV-2 and HDV-4 were fairly similar, albeit lower than that of HDV-1. Like HDV-1, both HDV-2 and HDV-4 showed a reduction in editing rate when the A.C mismatch pair and the immediately upstream base-paired region were disturbed. Notably, extending the downstream base-paired region from two to three or four (forming a structure identical to that of HDV-1) base pairs increased editing rate. Furthermore, we presented novel evidence that indicates the importance of the first bulge's size, located upstream of the editing site, and the base-pairing length within 7–13 and 28–39 nucleotides downstream of the editing site in influencing the HDV-4 editing rate. To summarize, our analyses suggest that the unbranched rod-like structures surrounding the editing site of HDV-2 and HDV-4 play a crucial role in regulating their RNA editing rates.

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.

Identification of Bona Fide RNA Editing Sites: History, Challenges, and Opportunities.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of enzymes of which there are three members (ADAR1, ADAR2, and ADAR3), is a major gene regulatory mechanism that diversifies the transcriptome. It is widespread in many metazoans, including humans. As inosine is interpreted by cellular machineries mainly as guanosine, A-to-I editing effectively gives A-to-G nucleotide changes. Depending on its location, an editing event can generate new protein isoforms or influence other RNA processing pathways. Researchers have found that ADAR-mediated editing performs diverse functions. For example, it enables living organisms such as cephalopods to adapt rapidly to fluctuating environmental conditions such as water temperature. In development, the loss of ADAR1 is embryonically lethal partly because endogenous double-stranded RNAs (dsRNAs) are no longer marked by inosines, which signal "self", and thus cause the melanoma differentiation-associated protein 5 (MDA5) sensor to trigger a deleterious interferon response. Hence, ADAR1 plays a key role in preventing aberrant activation of the innate immune system. Furthermore, ADAR enzymes have been implicated in myriad human diseases. Intriguingly, some cancer cells are known to exploit ADAR1 activity to dodge immune responses. However, the exact identities of immunogenic RNAs in different biological contexts have remained elusive. Consequently, there is tremendous interest in identifying inosine-containing RNAs in the cell.The identification of A-to-I RNA editing sites is dependent on the sequencing of nucleic acids. Technological and algorithmic advancements over the past decades have revolutionized the way editing events are detected. At the beginning, the discovery of editing sites relies on Sanger sequencing, a first-generation technology. Both RNA, which is reverse transcribed into complementary DNA (cDNA), and genomic DNA (gDNA) from the same source are analyzed. After sequence alignment, one would require an adenosine to be present in the genome but a guanosine to be detected in the RNA sample for a position to be declared as an editing site. However, an issue with Sanger sequencing is its low throughput. Subsequently, Illumina sequencing, a second-generation technology, was invented. By permitting the simultaneous interrogation of millions of molecules, it enables many editing sites to be identified rapidly. However, a key challenge is that the Illumina platform produces short sequencing reads that can be difficult to map accurately. To tackle the challenge, we and others developed computational workflows with a series of filters to discard sites that are likely to be false positives. When Illumina sequencing data sets are properly analyzed, A-to-G variants should emerge as the most dominant mismatch type. Moreover, the quantitative nature of the data allows us to build a comprehensive atlas of editing-level measurements across different biological contexts, providing deep insights into the spatiotemporal dynamics of RNA editing. However, difficulties remain in identifying true A-to-I editing sites in short protein-coding exons or in organisms and diseases where DNA mutations and genomic polymorphisms are prevalent and mostly unknown. Nanopore sequencing, a third-generation technology, promises to address the difficulties, as it allows native RNAs to be sequenced without conversion to cDNA, preserving base modifications that can be directly detected through machine learning. We recently demonstrated that nanopore sequencing could be used to identify A-to-I editing sites in native RNA directly. Although further work is needed to enhance the detection accuracy in single molecules from fewer cells, the nanopore technology holds the potential to revolutionize epitranscriptomic studies.

Assembly and comparative analysis of the complete mitochondrial genome of Viburnum chinshanense

BackgroundViburnum chinshanense is an endemic species found exclusively in the North-Central and South-Central regions of China. This species is a lush garden ornamental tree and is extensively utilized for vegetation restoration in rocky desertification areas.ResultsIn this study, we obtained 13.96 Gb of Oxford Nanopore data for the whole genome, and subsequently, by combining Illumina short-reads, we successfully assembled the complete mitochondrial genome (mitogenome) of the V. chinshanense using a hybrid assembly strategy. The assembled genome can be described as a circular genome. The total length of the V. chinshanense mitogenome measures 643,971 bp, with a GC content of 46.18%. Our annotation efforts have revealed a total of 39 protein-coding genes (PCGs), 28 tRNA genes, and 3 rRNA genes within the V. chinshanense mitogenome. The analysis of repeated elements has identified 212 SSRs, 19 long tandem repeat elements, and 325 pairs of dispersed repeats in the V. chinshanense mitogenome. Additionally, we have investigated mitochondrial plastid DNAs (MTPTs) and identified 21 MTPTs within the mitogenome and plastidial genome. These MTPTs collectively span a length of 9,902 bp, accounting for 1.54% of the mitogenome. Moreover, employing Deepred-mt, we have confidently predicted 623 C to U RNA editing sites across the 39 protein-coding genes. Furthermore, extensive genomic rearrangements have been observed between V. chinshanense and the mitogenomes of related species. Interestingly, we have also identified a bacterial-derived tRNA gene (trnC-GCA) in the V. chinshanense mitogenome. Lastly, we have inferred the phylogenetic relationships of V. chinshanense with other angiosperms based on mitochondrial PCGs.ConclusionsThis study marks the first report of a mitogenome from the Viburnum genus, offering a valuable genomic resource for exploring the evolution of mitogenomes within the Dipsacales order.

Assembly and comparative analysis of the complete mitochondrial genome of Isopyrum anemonoides (Ranunculaceae).

Ranunculaceae is a large family of angiosperms comprising 2500 known species-a few with medicinal and ornamental values. Despite this, only two mitochondrial genomes (mitogenomes) of the family have been released in GenBank. Isopyrum anemonoides is a medicinal plant belonging to the family Ranunculaceae, and its chloroplast genome has recently been reported; however, its mitogenome remains unexplored. In this study, we assembled and analyzed the complete mitochondrial genome of I. anemonoides and performed a comparative analysis against different Ranunculaceae species, reconstructing the phylogenetic framework of Isopyrum. The circular mitogenome of I. anemonoides has a length of 206,722 bp, with a nucleotide composition of A (26.4%), T (26.4%), C (23.6%), and G (23.6%), and contains 62 genes, comprising 37 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and three ribosomal RNA (rRNA) genes. Abundantly interspersed repetitive and simple sequence repeat (SSR) loci were detected in the I. anemonoides mitogenome, with tetranucleotide repeats accounting for the highest proportion of SSRs. By detecting gene migration, we observed gene exchange between the chloroplast and mitogenome in I. anemonoides, including six intact tRNA genes, six PCG fragments, and fragments from two rRNA genes. Comparative mitogenome analysis of three Ranunculaceae species indicated that the PCG contents were conserved and the GC contents were similar. Selective pressure analysis revealed that only two genes (nad1 and rpl5) were under positive selection during their evolution in Ranunculales, and two specific RNA editing sites (atp6 and mttB) were detected in the I. anemonoides mitogenome. Moreover, a phylogenetic analysis based on the mitogenomes of I. anemonoides and the other 15 taxa accurately reflected the evolutionary and taxonomic status of I. anemonoides. Overall, this study provides new insights into the genetics, systematics, and evolution of mitochondrial evolution in Ranunculaceae, particularly I. anemonoides.

FLARE: a fast and flexible workflow for identifying RNA editing foci

BackgroundFusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors.ResultsWe present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing.ConclusionsFLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available at https://github.com/YeoLab/FLARE.

Comparative chloroplast genome analysis of four Polygonatum species insights into DNA barcoding, evolution, and phylogeny

The Polygonatum genus represents a perennial herb with the Liliaceae family, boasting substantial economic and medicinal significance. The majority of Polygonatum plants exhibit notable similarity while lacking distinctive identifying characteristics, thus resulting in the proliferation of adulterated medicinal materials within the market. Within this study, we conducted an in-depth analysis of the complete chloroplast (cp) genomes of four Polygonatum plants and compared them with four closely akin species. The primary objectives were to unveil structural variations, species divergence, and the phylogenetic interrelations among taxa. The cp genomes of the four Polygonatum species were typified by a conventional quadripartite structure, incorporating a large single copy region (LSC), a small single copy region (SSC), and a pair of inverted repeat regions. In total, we annotated a range of 131 to 133 genes, encompassing 84 to 86 protein-coding genes, 38 transfer RNA (tRNA) genes, 8 ribosomal RNA (rRNA) genes, and 0 to 2 pseudogenes (ycf1, infA). Our comparative analyses unequivocally revealed a remarkable consistency in gene order and GC content within the Polygonatum genus. Furthermore, we predicted a potential 59 to 64 RNA editing sites distributed across 22 protein-coding genes, with the ndhB gene exhibiting the most prominent propensity for RNA editing sites, boasting a tally of 15 sites. Notably, six regions of substantial potential variability were ascertained, characterized by elevated Pi values. Noteworthy, molecular markers for species identification, population genetic scrutiny, and phylogenetic investigations within the genus were identified in the form of the psaJ-rpl33 and trnS + trnT-psaD barcodes. The resultant phylogenetic tree unequivocally depicted the formation of a monophyletic clade comprising species within the evolutionary framework of Liliaceae, demonstrating closer evolutionary affinities with Maianthemum, Dracaeneae, and Asparageae. This comprehensive compendium of findings collectively contributes to the advancement of molecular species identification, elucidation of phylogenetic interrelationships, and the establishment of DNA barcodes tailored to the Polygonatum species.

Genome-wide perturbations of A-to-I RNA editing dysregulated circular RNAs promoting the development of cervical cancer

Cervical cancer, the second most common female malignant tumor, seriously threatens women's health and lives. Despite the availability of the HPV vaccine, effective treatment options for cervical cancer are still lacking. New research perspectives now clarify that RNA editing dysregulation and changes in circRNA expression are jointly involved in disease pathogenesis, so molecular changes associated with circRNA and RNA editing may provide clues for the development of new therapeutic strategies for cervical cancer. In this study, we designed a series of pipelines to identify and analyze dysregulated RNA editing events in circRNAs. Our findings indicate a decrease in A-to-I RNA editing levels in cervical cancer compared to normal tissues, and editing may influence the back-splicing process of circRNAs through structural modifications of Alu elements. Moreover, our research reveals that RNA editing could modulate circRNA biogenesis by influencing RNA binding protein (RBP) binding on a transcriptome-wide scale, as well as influence the expression and coding potential of circRNAs. Importantly, we identified three RNA editing sites that could serve as potential biomarkers. In summary, our study presents a comprehensive landscape of RNA editing perturbations in circRNAs, providing new insights into the complex relationship between RNA editing and circRNA dysregulation in cervical cancer.

The Q/R editing site of AMPA receptor GluA2 subunit acts as an epigenetic switch regulating dendritic spines, neurodegeneration and cognitive deficits in Alzheimer’s disease

BackgroundRNA editing at the Q/R site of GluA2 occurs with ~99% efficiency in the healthy brain, so that the majority of AMPARs contain GluA2(R) instead of the exonically encoded GluA2(Q). Reduced Q/R site editing increases AMPA receptor calcium permeability and leads to dendritic spine loss, neurodegeneration, seizures and learning impairments. Furthermore, GluA2 Q/R site editing is impaired in Alzheimer’s disease (AD), raising the possibility that unedited GluA2(Q)-containing AMPARs contribute to synapse loss and neurodegeneration in AD. If true, then inhibiting expression of unedited GluA2(Q), while maintaining expression of GluA2(R), may be a novel strategy of preventing synapse loss and neurodegeneration in AD.MethodsWe engineered mice with the ‘edited’ arginine codon (CGG) in place of the unedited glutamine codon (CAG) at position 607 of the Gria2 gene. We crossbred this line with the J20 mouse model of AD and conducted anatomical, electrophysiological and behavioural assays to determine the impact of eliminating unedited GluA2(Q) expression on AD-related phenotypes.ResultsEliminating unedited GluA2(Q) expression in AD mice prevented dendritic spine loss and hippocampal CA1 neurodegeneration as well as improved working and reference memory in the radial arm maze. These phenotypes were improved independently of Aβ pathology and ongoing seizure susceptibility. Surprisingly, our data also revealed increased spine density in non-AD mice with exonically encoded GluA2(R) as compared to their wild-type littermates, suggesting an unexpected and previously unknown role for unedited GluA2(Q) in regulating dendritic spines.ConclusionThe Q/R editing site of the AMPA receptor subunit GluA2 may act as an epigenetic switch that regulates dendritic spines, neurodegeneration and memory deficits in AD.

Multi-Chromosomal mitochondrial genome of medicinal plant Acorus tatarinowii (Acoraceae): Firstly reported from Acorales Order

Acorus tatarinowii Schott (A. tatarinowii), a well-known traditional Chinese medicinal plant renowned for its high medicinal value, but its mitochondrial genome (mitogenome) is still unexplored. In this study, we meticulously assembled the complete mitochondrial genome of A. tatarinowii using a combination of Illumina short reads and Oxford Nanopore long reads. Our findings revealed that A. tatarinowii possesses a complex chromosomal structural mitogenome, comprising two linear chromosomes and seven circular chromosomes. This mitogenome spans 1.81 Mb in length with a GC content of 38.29 %. Notably, it contained 24 unique mitochondrial core genes, seven unique variable genes, 17 tRNA genes, and three rRNA genes. Analyses of codon usage, most protein-coding genes (PCGs) exhibited a common codon usage preference, with RSCU values greater than 1, and the codon with the highest RSCU value was UAA(End, 1.90). We conducted a thorough analysis of repeat sequences, the distribution of repetitive sequences in nine mitochondrial chromosomes showed distinct patterns. Moreover, we identified 82 and 12 homologous fragments by comparing the sequences of chloroplast and nuclear genomes to the A. tatarinowii mitogenome, respectively. Lastly, We predicted a total of 234 potential RNA editing sites in 28 unique PCGs and discovered that the nad4 gene has been edited the most often, at 26 times. Our results contribute to the enrichment of mitochondrial genome resources for Acoraceae, and the mitogenome also can be used as a reference for other species.

RNA editing catalytic complexes edit multiple mRNA sites non-processively in Trypanosoma brucei

RNA editing generates mature mitochondrial mRNAs in T. brucei by extensive uridine insertion and deletion at numerous editing sites (ESs) as specified by guide RNAs (gRNAs). The editing is performed by three RNA Editing Catalytic Complexes (RECCs) which each have a different endonuclease in addition to 12 proteins in common resulting in RECC1 that is specific for deletion ESs and RECC2 and RECC3 that are specific for insertion ESs. Thus, different RECCs are required for editing of mRNA sequence regions where single gRNAs specify a combination of insertion and deletion ESs. We investigated how the three different RECCs might edit combinations of insertion and deletion ESs that are specified by single gRNAs by testing whether their endonuclease compositions are stable or dynamic during editing. We analyzed in vivo BirA* proximity labeling and found that the endonucleases remain associated with their set of common RECC proteins during editing when expressed at normal physiological levels. We also found that overexpression of endonuclease components resulted in minor effects on RECCs but did not affect growth. Thus, the protein stoichiometries that exist within each RECC can be altered by perturbations of RECC expression levels. These results indicate that editing of consecutive insertion and deletion ESs occurs by successive engagement and disengagement of RECCs, i.e., is non-processive, which is likely the case for consecutive pairs of insertion or deletion ESs. This clarifies the nature of the complex patterns of partially edited mRNAs that occur in vivo.

Identification of microRNA editing sites in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor originating from the renal tubular epithelium. Although the microRNAs (miRNAs) transcriptome of ccRCC has been extensively studied, the role of miRNAs editing in ccRCC is largely unknown. By analyzing small RNA sequencing profiles of renal tissues of 154 ccRCC patients and 22 normal controls, we identified 1025 miRNA editing sites from 246 pre-miRNAs. There were 122 editing events with significantly different editing levels in ccRCC compared to normal samples, which include two A-to-I editing events in the seed regions of hsa-mir-376a-3p and hsa-mir-376c-3p, respectively, and one C-to-U editing event in the seed region of hsa-mir-29c-3p. After comparing the targets of the original and edited miRNAs, we found that hsa-mir-376a-1_49g, hsa-mir-376c_48g and hsa-mir-29c_59u had many new targets, respectively. Many of these new targets were deregulated in ccRCC, which might be related to the different editing levels of hsa-mir-376a-3p, hsa-mir-376c-3p, hsa-mir-29c-3p in ccRCC compared to normal controls. Our study sheds new light on miRNA editing events and their potential biological functions in ccRCC.

A full repertoire of Hemiptera genomes reveals a multi-step evolutionary trajectory of auto-RNA editing site in insect Adar gene

ABSTRACT Adenosine-to-inosine (A-to-I) RNA editing, mediated by metazoan ADAR enzymes, is a prevalent post-transcriptional modification that diversifies the proteome and promotes adaptive evolution of organisms. The Drosophila Adar gene has an auto-recoding site (termed S>G site) that forms a negative-feedback loop and stabilizes the global editing activity. However, the evolutionary trajectory of Adar S>G site in many other insects remains largely unknown, preventing us from a deeper understanding on the significance of this auto-editing mechanism. In this study, we retrieved the well-annotated genomes of 375 arthropod species including the five major insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera and Hemiptera) and several outgroup species. We performed comparative genomic analysis on the Adar auto-recoding S>G site. We found that the ancestral state of insect S>G site was an uneditable serine codon (unSer) and that this state was largely maintained in Hymenoptera. The editable serine codon (edSer) appeared in the common ancestor of Lepidoptera, Diptera and Coleoptera and was almost fixed in the three orders. Interestingly, Hemiptera species possessed comparable numbers of unSer and edSer codons, and a few ‘intermediate codons’, demonstrating a multi-step evolutionary trace from unSer-to-edSer with non-synchronized mutations at three codon positions. We argue that the evolution of Adar S>G site is the best genomic evidence supporting the ‘proteomic diversifying hypothesis’ of RNA editing. Our work deepens our understanding on the evolutionary significance of Adar auto-recoding site which stabilizes the global editing activity and controls transcriptomic diversity.

Benchmarking RNA Editing Detection Tools.

RNA, like DNA and proteins, can undergo modifications. To date, over 170 RNA modifications have been identified, leading to the emergence of a new research area known as epitranscriptomics. RNA editing is the most frequent RNA modification in mammalian transcriptomes, and two types have been identified: (1) the most frequent, adenosine to inosine (A-to-I); and (2) the less frequent, cysteine to uracil (C-to-U) RNA editing. Unlike other epitranscriptomic marks, RNA editing can be readily detected from RNA sequencing (RNA-seq) data without any chemical conversions of RNA before sequencing library preparation. Furthermore, analyzing RNA editing patterns from transcriptomic data provides an additional layer of information about the epitranscriptome. As the significance of epitranscriptomics, particularly RNA editing, gains recognition in various fields of biology and medicine, there is a growing interest in detecting RNA editing sites (RES) by analyzing RNA-seq data. To cope with this increased interest, several bioinformatic tools are available. However, each tool has its advantages and disadvantages, which makes the choice of the most appropriate tool for bench scientists and clinicians difficult. Here, we have benchmarked bioinformatic tools to detect RES from RNA-seq data. We provide a comprehensive view of each tool and its performance using previously published RNA-seq data to suggest recommendations on the most appropriate for utilization in future studies.