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

Viruses often pose a significant threat to the host through the exploitation of cellular machineries for their own benefit. In the context of immune responses, myriad host factors are deployed to target viral RNAs and inhibit viral protein translation, ultimately hampering viral replication. Understanding how \"non-self\" RNAs interact with the host translation machinery and trigger immune responses would help in the development of treatment strategies for viral infections. In this review, we explore how interferon-stimulated gene products interact with viral RNA and the translation machinery in order to induce either global or targeted translation inhibition.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2\'s preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes\' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

Adenosine-to-inosine (A-to-I) RNA editing recodes the genome and confers flexibility for the organisms to adapt to the environment. It is believed that RNA recoding sites are well suited for facilitating adaptive evolution by increasing the proteomic diversity in a temporal-spatial manner. The function and essentiality of a few conserved recoding sites are recognized. However, the experimentally discovered functional sites only make up a small corner of the total sites, and there is still the need to expand the repertoire of such functional sites with bioinformatic approaches. In this study, we define a new category of RNA editing sites termed \'conserved editing with non-conserved recoding\' and systematically identify such sites in Drosophila editomes, figuring out their selection pressure and signals of adaptation at inter-species and intra-species levels. Surprisingly, conserved editing sites with non-conserved recoding are not suppressed and are even slightly overrepresented in Drosophila. DNA mutations leading to such cases are also favoured during evolution, suggesting that the function of those recoding events in different species might be diverged, specialized, and maintained. Finally, structural prediction suggests that such recoding in potassium channel Shab might increase ion permeability and compensate the effect of low temperature. In conclusion, conserved editing with non-conserved recoding might be functional as well. Our study provides novel aspects in considering the adaptive evolution of RNA editing sites and meanwhile expands the candidates of functional recoding sites for future validation.

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.

Understanding phylogenetic relationships among species is essential for many biological studies, which call for an accurate phylogenetic tree to understand major evolutionary transitions. The phylogenetic analyses present a major challenge in estimation accuracy and computational efficiency, especially recently facing a wave of severe emerging infectious disease outbreaks. Here, we introduced a novel, efficient framework called Bases-dependent Rapid Phylogenetic Clustering (Bd-RPC) for new sample placement for viruses. In this study, a brand-new recoding method called Frequency Vector Recoding was implemented to approximate the phylogenetic distance, and the Phylogenetic Simulated Annealing Search algorithm was developed to match the recoded distance matrix with the phylogenetic tree. Meanwhile, the indel (insertion/deletion) was heuristically introduced to foreign sequence recognition for the first time. Here, we compared the Bd-RPC with the recent placement software (PAGAN2, EPA-ng, TreeBeST) and evaluated it in Alphacoronavirus, Alphaherpesvirinae, and Betacoronavirus by using Split and Robinson-Foulds distances. The comparisons showed that Bd-RPC maintained the highest precision with great efficiency, demonstrating good performance in new sample placement on all three virus genera. Finally, a user-friendly website (http://www.bd-rpc.xyz) is available for users to classify new samples instantly and facilitate exploration of the phylogenetic research in viruses, and the Bd-RPC is available on GitHub (http://github.com/Bin-Ma/bd-rpc).

Pseudoknots assume various functions including stimulation of -1 programmed ribosomal frameshifting (PRF) or stop codon readthrough (SCR) in RNA viruses. These pseudoknots vary greatly in sizes and structural complexities. Recent biochemical and structural studies confirm the three-stemmed pseudoknots as the -1 PRF stimulators in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related coronaviruses. We reexamined previously reported -1 PRF or SCR stimulating pseudoknots, especially those containing a relatively long connecting loop between the two pseudoknot-forming stems, for their ability to form elaborated structures. Many potential elaborated pseudoknots were identified that contain one or more of the following extra structural elements: stem-loop, embedded pseudoknot, kissing hairpins, and additional loop-loop interactions. The elaborated pseudoknots are found in several different virus families that utilize either the -1 PRF or SCR recoding mechanisms. Model-building studies were performed to not only establish the structural feasibility of the elaborated pseudoknots but also reveal potential additional structural features that cannot be readily inferred from the predicted secondary structures. Some of the structures, such as embedded double pseudoknots and compact loop-loop pseudoknots mediated by the previously established common pseudoknot motif-1 (CPK-1), represent the first of its kind in the literatures. By advancing discovery of new functional RNA structures, we significantly expand the repertoire of known elaborated pseudoknots that could potentially play a role in -1 PRF and SCR regulation. These results contribute to a better understanding of RNA structures in general, facilitating the design of engineering RNA molecules with certain desired functions.Communicated by Ramaswamy H. Sarma.

OBJECTIVE: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is one of the first reports that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

The adaptiveness of nonsynonymous RNA editing (recoding) could be conferred by the flexibility of the temporal-spatially controllable proteomic diversity, or by its restorative effect which fixes unfavorable genomic mutations at the RNA level. These two complementary hypotheses, namely, the diversifying hypothesis and the restorative hypothesis, have distinct predictions on the landscape of RNA editing sites. We collected the chloroplast C-to-U RNA editomes of 21 vascular plants (11 angiosperms, four gymnosperms, and six ferns) from a previous study, aiming to testify whether the plant editomes typically conform to the restorative hypothesis. All predictions made by the restorative hypothesis are verified: (i) nonsynonymous editing sites are more frequent and have higher editing levels than synonymous sites; (ii) nonsynonymous editing levels are extremely high and show weak tissue-specificity in plants; (iii) on the inferred genomic sites with recent T-to-C mutations, nonsynonymous sites but not synonymous sites are compensated by C-to-U RNA editing. In conclusion, nonsynonymous C-to-U RNA editing in plants is adaptive due to its restorative effects. The recoding levels are high and are constantly required across the whole plant so that the recoding events could perfectly mimic DNA mutations. The evolutionary significance of plant RNA editing is systematically demonstrated at the genome-wide level.

Experimental increase of cytosine-phosphate-guanine (CpG) dinucleotides in an RNA virus genome impairs infection. Beneficially, this weak infection may lead to robust antiviral host immunity providing a cutting-edge approach for vaccines. For example, we have recently demonstrated that recoded Zika virus variants with the increased CpG content showed considerable attenuated infection phenotypes and protection against lethal challenge in mice. Here, we describe the workflow for the design and generation of CpG-recoded Zika virus vaccine candidates. The workflow can be adapted for other viruses.