Editing Complex Research Articles

Site-selective C-H functionalization to access the arene backbone of indoles and quinolines.

The site-selective C-H bond functionalization of heteroarenes can eventually provide chemists with great techniques for editing and building complex molecular scaffolds. During the past decade, benzo-fused N-heterocycles such as indoles and quinolines have been among the most widely investigated organic templates. Early developments have led to site-selective C-H bond functionalization on the pyrrole and pyridine cores of indoles and quinolines; however, C-H functionalization on the benzenoid ring has remained a great challenge in catalysis. In this review, we elaborate on recent developments in the highly challenging functionalization of C-H bonds on the less-reactive benzenoid core of indoles and quinolines. These findings are mainly described as selective directing group assisted strategies, remote C-H functionalization techniques and their reaction mechanisms. The underlying principle in each strategy is elucidated, which aims to facilitate the design of a more advanced structure of heterocycles based on bioactive molecules, synthetic drugs, and material aspects. Moreover, the challenges and perspectives for catalytic C-H functionalization to access the arene backbone of indoles and quinolines are also proposed in the conclusion section.

Optimization of in vitro gene delivery to neurons containing an APP gene with a mutation responsible for familial Alzheimer’s disease for the development of a base editing therapy

AbstractBackgroundThe Amyloïd Precursor Protein (APP) is preferentially cut by the α‐secretase enzyme in healthy individuals. However, an abnormal cleavage by β‐secretase leads to the accumulation of β‐amyloid peptides. Numerous APP genetic mutations cause Familial Alzheimer Disease (FAD). However, a rare APP gene variant (A673T) found in Icelanders drastically reduces cleavage by the β‐secretase. We hypothesized that the insertion of this A673T mutation in a patient’s genome using the CRISPR/Cas9 Base‐Editing Technology (Komor et al. Nature 2016) could be an effective method to slow down the progression of familial and sporadic forms of Alzheimer’s disease. The objective of this study is to eventually compare the efficiency by which a lentiviral or a dual‐AAV‐based vector could deliver a base‐editing complex (SpCas9VQR‐Target‐AID) into neurons in vitro.MethodNeurons were obtained using two methods. The first method involved differentiating fibroblasts from a FAD patient or a mouse model with a London (V717I) mutation into neurons (Shrigley et al. JoVE 2018) (Figure 1), while the second method involved isolating hippocampal neurons from prenatal NL/F/G mice (Seibenhener et al. JoVE 2012). The dual AAV complex was designed to deliver two parts of the base editor gene separated by inteins (Villiger et al. Nat Med 2018). Hippocampal or induced neurons were transduced with lentivirus or AAV1. Cas9 detection was performed by immunofluorescence and Western Blot. The β‐amyloid peptides in supernatant were characterized and quantified by Meso Scale Discovery’s Aβ kit.ResultWe have been able to detect the Cas9 protein in neurons by western blot and immunofluorescence (Figure 2). In subsequent experiments, we will determine which delivery method will reduce more the β‐amyloid peptide.ConclusionOur approach aims to confirm the protective effect of the A673T mutation against the development of familial and sporadic forms of Alzheimer’s disease as well as develop an efficient delivery method for the base editing complex which will lead to an in vivo application. Long‐term perspective: We aim to develop a completely new therapeutic approach for AD and FAD using base editing or the new PRIME editing technique (Anzalone et al. Nature 2019). This may eventually lead to a Phase I clinical trial.

Delivery of CRISPR/Cas9 Ribonucleoprotein Complex into Plant Apical Meristem Cells Leads to Large Deletions in an Editing Gene

Genome editing using the CRISPR/Cas9 system is an innovation platform exploiting site-specific nuclease to create modifications (deletions or insertions) into a target region of the genome of eukaryotic organisms. Plasmid-free methods of editing via delivery of the preformed RNP complex consisting of Cas 9 endonuclease and short guide RNA are of particular interest in modern plant biology. The use of these methods rules out an integration of foreign DNA into the plant genome. We have recently developed the method of genome editing for the potato crop using delivery of the RNP editing complex immobilized on gold or chitosan microparticles into meristem cells. Here, using the gene of coilin, the structural protein of Cajal bodies which also controls plant stress resistance, we show that this method leads to the formation of large deletions of about 500 bp in the gene, which are not common for the CRISPR/Cas9 system possibly due to molecular-genetic peculiarities of meristem cells. Data presented are discussed in the context of practical applications of this method.

High APOBEC1 Complementation Factor Expression Positively Modulates the Proliferation, Invasion, and Migration of Endometrial Cancer Cells Through Regulating P53/P21 Signaling Pathway.

Background: APOBEC1 complementation factor (A1CF) is a component of the apolipoprotein-B messenger RNA editing complex that participates in various cellular processes and acts as an oncogene in many cancers. In this study, it was aimed to investigate the roles of A1CF and its potential mechanism in endometrial cancer (EC). Materials and Methods: Gene expression prolife was downloaded from The Cancer Genome Atlas database. Then Kaplan-Meier and Cox regression analyses were conducted to assess the prognostic value of A1CF in EC. Cell Counting Kit-8, plate clone formation, and transwell assays were used to estimate the functions of A1CF on the proliferation, invasion, and migration of EC cell. The gene set enrichment analysis was used to analyze the pathway that is enriched by A1CF, whereas quantitative real-time polymerase chain reaction and Western blot analyses were utilized to detect the mRNA and protein expression involved. Results: It was detected that the upregulated A1CF was enriched in P53/P21 signaling pathway and tightly associated with patients' age, stage, and death. Besides, high A1CF expression led to a shorter overall survival of patients and predicted a poor prognosis in EC. The overexpression of A1CF promoted the proliferation, invasion, and migration of EC cells, whereas the depletion of A1CF suppressed these processes. Moreover, P21 and P53 were reduced whereas cyclin D1 and proliferating cell nuclear antigen were induced along with the increasing of A1CF. However, the effects of silencing A1CF on these protein expressions were on the contrary. Conclusion: A1CF was highly expressed and closely related to the prognosis and progression of EC through the regulation of P53/P21 signaling pathway, providing a possible new therapy target site for EC.

Gene editing particle system as a therapeutic approach for drug-resistant colorectal cancer

The epidermal growth factor receptor (EGFR) pathway plays an important role in the progression of colorectal cancer (CRC). Anti-EGFR drugs based on antibodies have been widely used for treating CRC through inducing the cell death pathway. However, the majority of CRC patients will inevitably develop drug-resistance during anti-EGFR drug treatment, which is mainly caused by a point mutation in the KRAS oncogene. We developed a nanoliposomal (NL) particle containing the Cas9 protein and a single-guide RNA (sgRNA) complex (Cas9-RNP), for genomic editing of the KRAS mutation. The NL particle is composed of bio-compatible lipid compounds that can effectively encapsulate Cas9-RNP. By modifying the NL particle to include the appropriate antibody, it can specifically recognize EGFR expressing CRC and effectively deliver the gene-editing complexes. The conditions of NL treatment were optimized using a KRAS mutated CRC in vivo mouse model. Mice with KRAS-mutated CRC showed drug resistance against cetuximab, a therapeutic antibody drug. After treating the mice with the KRAS gene-editing NL particles, the implanted tumors showed a dramatic decrease in size. Our results demonstrated that this genomic editing complex has great potential as a therapeutic carrier system for the treatment of drug-resistant cancer caused by a point mutation.

What is the Functional Role of Organelle Zinc finger 1 in the Plant C‐to‐U RNA Editing Complex?

Higher plants utilize protein complexes to precisely edit hundreds of RNA transcripts in organelles. The mechanistic model of RNA editing involves a C‐to‐U deamination reaction facilitated through the interaction of the RNA with a protein complex. Complex models have been proposed, but the minimal “editosome” capable of catalyzing C‐to‐U changes and the functions of many of the protein components have not been revealed. This research focuses on describing the functional role of one critical protein component belonging to the RAN binding protein 2 type zinc fingers protein family, Organelle Zinc finger 1 (OZ1). Zinc finger proteins have been associated with RNA binding; though RNA binding and site recognition in editosomes is known to occur via PPR proteins, OZ1 may be interacting with a scaffold RNA sequence. Recombinant OZ1 from Arabidopsis thaliana was used to analyze zinc coordination and RNA binding of this zinc finger protein. Spectrophotometric analysis of OZ1 after immobilized metal affinity chromatography (IMAC) suggests nucleic acid association. ICP‐MS and ESI‐MS analysis of a truncated variant of OZ1, only the sequence containing its two zinc finger domains (OZ1zf), suggests that OZ1 is coordinating only one zinc ion instead of the proposed two. Computational analysis of OZ1 dependent sites in Brachypodium distachyon returned a potential motif conserved among OZ1‐dependent editing sites. Electrophoretic mobility shift assays (EMSAs) are used to test RNA binding and the motif. Understanding this protein will bring us a step closer to manipulating this precise editing machine for use in biotechnology and may be used for purposes of crop improvement.Support or Funding InformationFunding for this work was provided by NIH SCORE SC2 ID: SC2 GM122718 awarded to Dr. Michael L. Hayes and the NIH MBRS‐RISE R25 GM 061331‐17 grant in support of Alfredo Jimenez‐Salinas.

Novel Interactions of Adrenodoxin-Related [2Fe-2S] Plant Ferredoxins MFDX1 and MFDX2 Indicate Their Involvement in a Wide Spectrum of Functions in Plant Mitochondria

Abstract Electron transfer chains of plant organelles (both chloroplasts and mitochondria) contain their own special set of ferredoxins. The relatively recently described adrenodoxin-like [2Fe-2S]-ferredoxins MFDX1 and MFDX2 of plant mitochondria are among the least studied of these. Until now, the only established function for them is participation in the final stage of biotin biosynthesis. In this work, using genetic and biochemical approaches, we searched for possible partners of these proteins in the genomes and proteomes of tobacco (Nicotiana tabacum L.) and foxglove (Digitalis purpurea L.) plants. MORF9 protein, one of the auxiliary components of the RNA editing complex of organelles (editosome), was found among the most prominent protein partners of adrenodoxin-like [2Fe-2S] tobacco ferredoxins. According to the results obtained from the yeast two-hybrid system, NtMFDX1 and NtMFDX2 of tobacco also bind and interact productively with the previously uncharacterised long non-coding polyadenylated RNA, which, based on its structural features, is capable of regulating the function of a number of components of complexes I (Nad1, Nad5) and III (protein of the cytochrome c synthesis system CcmF) and contributes to the formation of Fe/S-clusters in the corresponding protein complexes of the respiratory chain of plant mitochondria. We found one of the main components of the thiazol synthase complex (mitochondrial protein DpTHI1) to be the partner of ferredoxin DpMFDX2 of Digitalis purpurea. Finally, additional arguments were obtained in favour of the possible participation of MFDX1 and MFDX2 in the very ancient, but only recently described ‘progesterone’ steroid hormonal regulatory system: in leaves of the previously constructed CYP11A1-transgenic tomato plants, only the mature form of mitochondrial cytochrome P450scc (CYP11A1) of mammals is able to enter the mitochondria, where the above-mentioned components of the electron transport chain are localised. In summary, all of the newly revealed interactions of adrenodoxin-like [2Fe-2S] ferredoxins MFDX1 and MFDX2 indicate their participation in a wide range of functions in plant mitochondria.

Analysis of Tissue-specific RNA Editing Events of Genes Involved in RNA Editing in Arabidopsis thaliana

In plants, RNA editing is a post-transcriptional modification of transcripts and commonly occurs in plastids and mitochondria. In the case of flowering plants, not only PPR but also non-PPR proteins, such as MORF/RIP, ORRM and OZ partake in the diverse RNA editing complex. In Arabidopsis thaliana, 12 types of RNA editing patterns have been predicted in nuclear, chloroplast, and mitochondrial transcripts. In this study, tissue-specific RNA editing events were detected in gene families involved in RNA editing. Different Arabidopsis tissues at variable developmental stages, including 4-, 8-, and 12-d-old seedlings and 16-, 21-, 27-, and 32-d-old leaves, stems, stipes and roots, were collected and used for this study. Nine types of RNA editing events, including C-to-U, U-to-C, A-to-I, A-to-C, A-to-U, G-to-A, G-to-C, U-to-A and U-to-G, were identified in target genes. Most of the editing events occurred in seedlings and leaves and a few in stem tissues. Extensive U-to-C editing (60%) and A-to-G editing (54%) was detected in 12-d-old seedlings and 21-d-old leaves, respectively. This is the first experimental report of tissue-specific nuclear RNA editing events found in plants. This study will provide important information for revealing the mechanism of RNA editing in plants.

MORF9 Functions in Plastid RNA Editing with Tissue Specificity.

RNA editing in plant mitochondria and plastids converts specific nucleotides from cytidine (C) to uridine (U). These editing events differ among plant species and are relevant to developmental stages or are impacted by environmental conditions. Proteins of the MORF family are essential components of plant editosomes. One of the members, MORF9, is considered the core protein of the editing complex and is involved in the editing of most sites in chloroplasts. In this study, the phenotypes of a T-DNA insertion line with loss of MORF9 and of the genetic complementation line of Arabidopsis were analyzed, and the editing efficiencies of plastid RNAs in roots, rosette leaves, and flowers from the morf9 mutant and the wild-type (WT) control were compared by bulk-cDNA sequencing. The results showed that most of the known MORF9-associated plastid RNA editing events in rosette leaves and flowers were similarly reduced by morf9 mutation, with the exception that the editing rate of the sites ndhB-872 and psbF-65 declined in the leaves and that of ndhB-586 decreased only in the flowers. In the roots, however, the loss of MORF9 had a much lower effect on overall plastid RNA editing, with nine sites showing no significant editing efficiency change, including accD-794, ndhD-383, psbZ-50, ndhF-290, ndhD-878, matK-706, clpP1-559, rpoA-200, and ndhD-674, which were reduced in the other tissues. Furthermore, we found that during plant aging, MORF9 mRNA level, but not the protein level, was downregulated in senescent leaves. On the basis of these observations, we suggest that MORF9-mediated RNA editing is tissue-dependent and the resultant organelle proteomes are pertinent to the specific tissue functions.

Mutational processes contributing to the development of multiple myeloma

To gain insight into multiple myeloma (MM) tumorigenesis, we analyzed the mutational signatures in 874 whole-exome and 850 whole-genome data from the CoMMpass Study. We identified that coding and non-coding regions are differentially dominated by distinct single-nucleotide variant (SNV) mutational signatures, as well as five de novo structural rearrangement signatures. Mutational signatures reflective of different principle mutational processes—aging, defective DNA repair, and apolipoprotein B editing complex (APOBEC)/activation-induced deaminase activity—characterize MM. These mutational signatures show evidence of subgroup specificity—APOBEC-attributed signatures associated with MAF translocation t(14;16) and t(14;20) MM; potentially DNA repair deficiency with t(11;14) and t(4;14); and aging with hyperdiploidy. Mutational signatures beyond that associated with APOBEC are independent of established prognostic markers and appear to have relevance to predicting high-risk MM.

Evaluation and Reduction of CRISPR Off-Target Cleavage Events.

Introduction of CRISPR/Cas9 methods (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) have led to a huge surge in the use of precision genome editing for research applications. Translational medical efforts are likewise rapidly progressing, and Phase I clinical trials using these techniques have already started. As with any new technology that is applied to medical therapeutics, risks must be carefully defined and steps taken to mitigate side effects wherever possible. Effective methods are now available that permit identification of off-target cleavage events, a major class of potential side effects seen in mammalian genome editing. Off-target prediction algorithms are improving and have utility, but are insufficient to use alone. Empiric methods to define the off-target profile must also be used. Once defined, the frequency of off-target cleavage can be minimized using methods that limit the duration of exposure of the genome to the active genome editing complex, for example, using the ribonucleoprotein (RNP) approach. In addition, Cas9 mutants have been developed that markedly reduce the rate of off-target cleavage compared to the wild-type enzyme. Use of these new tools should become standard practice for medical applications.

Integrative genomic analysis identifies associations of molecular alterations to APOBEC and BRCA1/2 mutational signatures in breast cancer.

BackgroundThe observed mutations in cancer are the result of ~30 mutational processes, which stamp particular mutational signatures (MS). Nevertheless, it is still not clear which genomic alterations correlate to several MS. Here, a method to analyze associations of genomic data with MS is presented and applied to The Cancer Genome Atlas breast cancer data revealing promising associations.MethodsThe MS were discretized into clusters whose extremes were statistically associated with mutations, copy number, and gene expression data.ResultsKnown associations for apolipoprotein B editing complex (APOBEC) and for BRCA1 and BRCA2 support the proposal. For BRCA1/2, mutations in ARAP3, three focal deletions, and one amplification were detected. Around 50 mutated genes for the two APOBEC signatures were identified including three kinesins (KIF13A, KIF1B, KIF4A), three ubiquitins (USP45, UBR4, UBR1), and two demethylases (KDM5B, KDM5C) among other genes also connected to DNA damage pathways. The results suggest novel roles for other genes currently not involved in DNA repair. The altered expression program was very high for the BRCA1/2 signature, high for APOBEC signature 13 clearly associated to immune response, and low for APOBEC signature 2. The remaining signatures show scarce associations.ConclusionSpecific genetic alterations can be associated with particular MS.

SMK6 mediates the C-to-U editing at multiple sites in maize mitochondria

The recently identified PPR-E+/NVWA/DYW2 RNA editing complex provides insights into the mechanism of RNA editing in higher plant organelles. However, whether the complex works together with the previously identified editing factors RIPs/MORFs is unclear. In this paper, we identified a maize Smk6 gene, which encodes a mitochondrion-targeted PPR-E+protein with E1 and E2 domains at the C terminus. Loss of Smk6 function affects the C-to-U editing at nad1-740, nad4L-110, nad7-739, and mttB-138,139 sites, impairs mitochondrial activity and blocks embryogenesis and endosperm development. Genetic and molecular analysis indicated that SMK6 is the maize ortholog of the Arabidopsis SLO2, which is a component of the PPR-E+/NVWA/DYW2 editing complex. However, yeast two-hybrid analyses did not detect any interaction between SMK6 and any of the mitochondrion-targeted RIPs/MORFs, suggesting that RIPs/MORFs may not be a component of PPR-E+/NVWA/DYW2 RNA editing complex. Further analyses are required to provide evidence that RIP/MORFs and SMK6 do not physically interact in vivo.

CRISPR V Culture

New gene editing technologies give us the potential ability to bring back extinct species, help control the spread of invasive ones, and genetically modify those that spread diseases. They allows us to not only influence the evolutionary path of entire species, but entire ecosystems as well. Furthermore, gene editing has the potential to help us live healthier and longer lives. We have moved past rudimentary macroscopic methods of DNA manipulation and can now remove individual genes from a strand of DNA. However, due to the complexity of this technology, and given that there are few who can use it to its full effect, people have largely failed to respond to its development, particularly regulators. It is not within the scope of this paper to explore the full implications of these various emerging technologies, so instead I will focus on CRISPR, a specific new gene editing complex first used in 2012, and the major developments that have taken place since then.

The RNA binding activity of the first identified trypanosome protein with Z-DNA-binding domains

RNA-binding proteins play a particularly important role in regulating gene expression in trypanosomes. A map of the network of protein complexes in Trypanosoma brucei uncovered an essential protein (Tb927.10.7910) that is postulated to be an RNA-binding protein implicated in the regulation of the mitochondrial post-transcriptional gene regulatory network by its association with proteins that participate in a multi-protein RNA editing complex. However, the mechanism by which this protein interacts with its multiple target transcripts remained unknown. Using sensitive database searches and experimental data, we identify Z-DNA-binding domains in T. brucei in the N- and C-terminal regions of Tb927.10.7910. RNA-binding studies of the wild-type protein, now referred to as RBP7910 (RNA binding protein 7910), and site-directed mutagenesis of residues important for the Z-DNA binding domains show that it preferentially interacts with RNA molecules containing poly(U) and poly(AU)-rich sequences. The interaction of RBP7910 with these regions may be involved in regulation of RNA editing of mitochondrial transcripts.

Identification of Targetable Lesions in Anaplastic Thyroid Cancer by Genome Profiling.

Anaplastic thyroid cancer (ATC) is a rare and extremely malignant tumor with no available cure. The genetic landscape of this malignancy has not yet been fully explored. In this study, we performed whole exome sequencing and the RNA-sequencing of fourteen cases of ATC to delineate copy number changes, fusion gene events, and somatic mutations. A high frequency of genomic amplifications was seen, including 29% of cases having amplification of CCNE1 and 9% of CDK6; these events may be targetable by cyclin dependent kinase (CDK) inhibition. Furthermore, 9% harbored amplification of TWIST1, which is also a potentially targetable lesion. A total of 21 fusion genes in five cases were seen, none of which were recurrent. Frequent mutations included TP53 (55%), the TERT promoter (36%), and ATM (27%). Analyses of mutational signatures showed an involvement of processes that are associated with normal aging, defective DNA mismatch repair, activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity, failure of DNA double-strand break repair, and tobacco exposure. Taken together, our results shed new light on the tumorigenesis of ATC and show that a relatively large proportion (36%) of ATCs harbor genetic events that make them candidates for novel therapeutic approaches. When considering that ATC today has a mortality rate of close to 100%, this is highly relevant from a clinical perspective.

Comparison of RNA Editing Activity of APOBEC1-A1CF and APOBEC1-RBM47 Complexes Reconstituted in HEK293T Cells

RNA editing is an important form of regulating gene expression and activity. APOBEC1 cytosine deaminase was initially characterized as pairing with a cofactor, A1CF, to form an active RNA editing complex that specifically targets APOB RNA in regulating lipid metabolism. Recent studies revealed that APOBEC1 may be involved in editing other potential RNA targets in a tissue-specific manner, and another protein, RBM47, appears to instead be the main cofactor of APOBEC1 for editing APOB RNA. In this report, by expressing APOBEC1 with either A1CF or RBM47 from human or mouse in an HEK293T cell line with no intrinsic APOBEC1/A1CF/RBM47 expression, we have compared direct RNA editing activity on several known cellular target RNAs. By using a sensitive cell-based fluorescence assay that enables comparative quantification of RNA editing through subcellular localization changes of eGFP, the two APOBEC1 cofactors, A1CF and RBM47, showed clear differences for editing activity on APOB and several other tested RNAs, and clear differences were observed when mouse versus human genes were tested. In addition, we have determined the minimal domain requirement of RBM47 needed for activity. These results provide useful functional characterization of RBM47 and direct biochemical evidence for the differential editing selectivity on a number of RNA targets.

Application of the CRISPR/Cas System for Generation of Pathogen-Resistant Plants.

The use of the CRISPR/Cas9 prokaryotic adaptive immune system has led to a breakthrough in targeted genome editing in eukaryotes. The CRISPR/Cas technology allows to generate organisms with desirable characteristics by introducing deletions/insertions into selected genome loci resulting in the knockout or modification of target genes. This review focuses on the current state of the CRISPR/Cas use for the generation of plants resistant to viruses, bacteria, and parasitic fungi. Resistance to DNA- and RNA-containing viruses is usually provided by expression in transgenic plants of the Cas endonuclease gene and short guide RNAs (sgRNAs) targeting certain sites in the viral or the host plant genomes to ensure either direct cleavage of the viral genome or modification of the plant host genome in order to decrease the efficiency of virus replication. Editing of plant genes involved in the defense response to pathogens increases plants resistance to bacteria and pathogenic fungi. The review explores strategies and prospects of the development of pathogen-resistant plants with a focus on the generation of non-transgenic (non-genetically modified) organisms, in particular, by using plasmid (DNA)-free systems for delivery of the Cas/sgRNA editing complex into plant cells.

Stoichiometry of triple-sieve tRNA editing complex ensures fidelity of aminoacyl-tRNA formation.

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids onto tRNAs. To avoid mistranslation, editing mechanisms evolved to maintain tRNA aminoacylation fidelity. For instance, while rejecting the majority of non-cognate amino acids via discrimination in the synthetic active site, prolyl-tRNA synthetase (ProRS) misactivates and mischarges Ala and Cys, which are similar in size to cognate Pro. Ala-tRNAPro is specifically hydrolyzed by the editing domain of ProRS in cis, while YbaK, a free-standing editing domain, clears Cys-tRNAPro in trans. ProXp-ala is another editing domain that clears Ala-tRNAPro in trans. YbaK does not appear to possess tRNA specificity, readily deacylating Cys-tRNACysin vitro. We hypothesize that YbaK binds to ProRS to gain specificity for Cys-tRNAPro and avoid deacylation of Cys-tRNACys in the cell. Here, in vivo evidence for ProRS-YbaK interaction was obtained using a split-green fluorescent protein assay. Analytical ultracentrifugation and native mass spectrometry were used to investigate binary and ternary complex formation between ProRS, YbaK, and tRNAPro. Our combined results support the hypothesis that the specificity of YbaK toward Cys-tRNAPro is determined by the formation of a three-component complex with ProRS and tRNAPro and establish the stoichiometry of a ‘triple-sieve’ editing complex for the first time.

AID/APOBEC-like cytidine deaminases are ancient innate immune mediators in invertebrates

In the course of both innate and adaptive immunity, cytidine deaminases within the activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) family modulate immune responses by mutating specific nucleic acid sequences of hosts and pathogens. The evolutionary emergence of these mediators, however, seems to coincide precisely with the emergence of adaptive immunity in vertebrates. Here, we show a family of genes in species within two divergent invertebrate phyla—the echinoderm Strongylocentrotus purpuratus and the brachiopod Lingula anatina—that encode proteins with similarities in amino acid sequence and enzymatic activities to the vertebrate AID/APOBECs. The expression of these invertebrate factors is enriched in tissues undergoing constant, direct interactions with microbes and can be induced upon pathogen challenge. Our findings suggest that AID/APOBEC proteins, and their function in immunity, emerged far earlier than previously thought. Thus, cytidine deamination is probably an ancient innate immune mechanism that predates the protostome/deuterostome divergence.