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Adar-mediated adenosine-to-inosine (A-to-I) mRNA editing is a conserved mechanism that exerts diverse regulatory functions during the development, evolution, and adaptation of metazoans. The accurate detection of RNA editing sites helps us understand their biological significance. In this work, with an improved genome assembly of honeybee (Apis mellifera), we used a new orthology-based methodology to complement the traditional pipeline of (de novo) RNA editing detection. Compared to the outcome of traditional pipeline, we retrieved many novel editing sites in CDS that are deeply conserved between honeybee and other distantly related insects. The newly retrieved sites were missed by the traditional de novo identification due to the stringent criteria for controlling false-positive rate. Caste-specific editing sites are identified, including an Ile>Met auto-recoding site in Adar. This recoding was even conserved between honeybee and bumblebee, suggesting its putative regulatory role in shaping the phenotypic plasticity of eusocial Hymenoptera. In summary, we proposed a complementary approach to the traditional pipeline and retrieved several previously unnoticed CDS editing sites. From both technical and biological aspects, our works facilitate future researches on finding the functional editing sites and advance our understanding on the connection between RNA editing and the great phenotypic diversity of organisms.

Abstract 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.

ABSTRACTAlthough A-to-I RNA editing leads to similar effects to A-to-G DNA mutation, nonsynonymous RNA editing (recoding) is believed to confer its adaptiveness by ‘epigenetically’ regulating proteomic diversity in a temporospatial manner, avoiding the pleiotropic effect of genomic mutations. Recent discoveries on the evolutionary trajectory of Ser>Gly auto-editing site in insect Adar gene demonstrated a selective advantage to having an editable codon compared to uneditable ones. However, apart from pure observations, quantitative approaches for justifying the adaptiveness of individual RNA editing sites are still lacking. We performed a comparative genomic analysis on 113 Diptera species, focusing on the Adar Ser>Gly auto-recoding site in Drosophila. We only found one species having a derived Gly at the corresponding site, and this occurrence was significantly lower than genome-wide random expectation. This suggests that the Adar Ser>Gly site is unlikely to be genomically replaced with G during evolution, and thus indicating the advantage of editable status over hardwired genomic alleles. Similar trends were observed for the conserved Ile>Met recoding in gene Syt1. In the light of evolution, we established a comparative genomic approach for quantitatively justifying the adaptiveness of individual editing sites. Priority should be given to such adaptive editing sites in future functional studies.

ABSTRACTAdar-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.

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.

Adenosine-to-inosine (A-to-I) editing is hypothesized to facilitate adaptive evolution by expanding proteomic diversity through an epigenetic approach. However, it is challenging to provide evidences to support this hypothesis at the whole editome level. In this study, we systematically characterized 2,114 A-to-I RNA editing sites in female and male brains of D. melanogaster, and nearly half of these sites had events evolutionarily conserved across Drosophila species. We detected strong signatures of positive selection on the nonsynonymous editing sites in Drosophila brains, and the beneficial editing sites were significantly enriched in genes related to chemical and electrical neurotransmission. The signal of adaptation was even more pronounced for the editing sites located in X chromosome or for those commonly observed across Drosophila species. We identified a set of gene candidates (termed "PSEB" genes) that had nonsynonymous editing events favored by natural selection. We presented evidence that editing preferentially increased mutation sequence space of evolutionarily conserved genes, which supported the adaptive evolution hypothesis of editing. We found prevalent nonsynonymous editing sites that were favored by natural selection in female and male adults from five strains of D. melanogaster. We showed that temperature played a more important role than gender effect in shaping the editing levels, although the effect of temperature is relatively weaker compared to that of species effect. We also explored the relevant factors that shape the selective patterns of the global editomes. Altogether we demonstrated that abundant nonsynonymous editing sites in Drosophila brains were adaptive and maintained by natural selection during evolution. Our results shed new light on the evolutionary principles and functional consequences of RNA editing.