Adenosine Deaminases Acting On RNA Research Articles

Characterization of tumor microenvironment and tumor immunology based on the double-stranded RNA-binding protein related genes in cervical cancer

BackgroundCervical cancer is one of the most common gynecological cancers threatening women’s health worldwide. Double-stranded RNA-binding proteins (dsRBPs) regulate innate immunity and are therefore believed to be involved in virus-related malignancies, however, their role in cervical cancer is not well known.MethodsWe performed RNA-seq of tumor samples from cervical cancer patients in local cohort and also assessed the RNA-seq and clinical data derived from public datasets. By using single sample Gene Set Enrichment Analysis (ssGSEA) and univariate Cox analysis, patients were stratified into distinct dsRBP clusters. Stepwise Cox and CoxBoost were performed to construct a risk model based on optimal dsRBPs clusters-related differentially expressed genes (DEGs), and GSE44001 and CGCI-HTMCP-CC were employed as two external validation cohorts. Single cell RNA sequencing data from GSE168652 and Scissor algorithm were applied to evaluated the signature-related cell population.ResultsThe expression of dsRBP features was found to be associated with HPV infection and carcinogenesis in CESC. However, only Adenosine deaminases acting on RNA (ADAR) and Dicer, Drosha, and Argonautes (DDR) exhibited significant correlations with the overall survival (OS) of CESC patients. Based on these findings, CESC patients were divided into three dsRBP clusters. Cluster 3 showed superior OS but lower levels of ADAR and DDR. Additionally, Cluster 3 demonstrated enhanced innate immunity, with significantly higher activity in cancer immunity cycles, immune scores, and levels of tumor-infiltrating immune cells, particularly CD8+ T cells. Furthermore, a risk model based on nine dsRBP cluster-related DEGs was established. The accuracy of survival prediction for 1 to 5 years was consistently above 0.78, and this model’s robust predictive capacity was confirmed by two external validation sets. The low-risk group exhibited significantly higher levels of immune checkpoints, such as PDCD1 and CTLA4, as well as a higher abundance of CD8+ T cells. Analysis of single-cell sequencing data revealed a significant association between the dsRBP signature and glycolysis. Importantly, low-risk patients showed improved OS and a higher response rate to immunotherapy, along with enduring clinical benefits from concurrent chemoradiotherapy.ConclusionsdsRBP played a crucial role in the regulation of prognosis and tumor immunology in cervical cancer, and its prognostic signature provides a strategy for risk stratification and immunotherapy evaluation.

High-level RNA editing diversifies the coleoid cephalopod brain proteome.

Coleoid cephalopods (octopus, squid and cuttlefish) have unusually complex nervous systems. The coleoid nervous system is also the only one currently known to recode the majority of expressed proteins through A-to-I RNA editing. The deamination of adenosine by adenosine deaminase acting on RNA (ADAR) enzymes produces inosine, which is interpreted as guanosine during translation. If this occurs in an open reading frame, which is the case for tens of thousands of editing sites in coleoids, it can recode the encoded protein. Here, we describe recent findings aimed at deciphering the mechanisms underlying high-level recoding and its adaptive potential. We describe the complement of ADAR enzymes in cephalopods, including a recently discovered novel domain in sqADAR1. We further summarize current evidence supporting an adaptive role of high-level RNA recoding in coleoids, and review recent studies showing that a large proportion of recoding sites is temperature-sensitive. Despite these new findings, the mechanisms governing the high level of RNA recoding in coleoid cephalopods remain poorly understood. Recent advances using genome editing in squid may provide useful tools to further study A-to-I RNA editing in these animals.

Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System.

Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA.

RNA Overwriting of Cellular mRNA by Cas13b-Directed RNA-Dependent RNA Polymerase of Influenza A Virus.

Dysregulation of mRNA processing results in diseases such as cancer. Although RNA editing technologies attract attention as gene therapy for repairing aberrant mRNA, substantial sequence defects arising from mis-splicing cannot be corrected by existing techniques using adenosine deaminase acting on RNA (ADAR) due to the limitation of adenosine-to-inosine point conversion. Here, we report an RNA editing technology called "RNA overwriting" that overwrites the sequence downstream of a designated site on the target RNA by utilizing the RNA-dependent RNA polymerase (RdRp) of the influenza A virus. To enable RNA overwriting within living cells, we developed a modified RdRp by introducing H357A and E361A mutations in the polymerase basic 2 of RdRp and fusing the C-terminus with catalytically inactive Cas13b (dCas13b). The modified RdRp knocked down 46% of the target mRNA and further overwrote 21% of the mRNA. RNA overwriting is a versatile editing technique that can perform various modifications, including addition, deletion, and mutation introduction, and thus allow for repair of the aberrant mRNA produced by dysregulation of mRNA processing, such as mis-splicing.

Emergence of biased hypermutation in a heterologous additional transcription unit in recombinant lentogenic Newcastle disease virus.

Recombinant Newcastle disease virus (rNDV) strains engineered to express foreign genes from an additional transcription unit (ATU) are considered as candidate live-attenuated vector vaccines for human and veterinary use. Early during the COVID-19 pandemic we and others generated COVID-19 vaccine candidates based on rNDV expressing a partial or complete SARS-CoV-2 spike (S) protein. In our studies, a number of the rNDV constructs did not show high S expression levels in cell culture or seroconversion in immunized hamsters. Sanger sequencing showed the presence of frequent A-to-G transitions characteristic of adenosine deaminase acting on RNA (ADAR). Subsequent whole genome rNDV sequencing revealed that this biased hypermutation was exclusively localized in the ATU expressing the spike gene, and was related to deamination of adenosines in the negative strand viral genome RNA. The biased hypermutation was found both after virus rescue in chicken cell line DF-1 followed by passaging in embryonated chicken eggs, and after direct virus rescue and subsequent passaging in Vero E6 cells. Levels of biased hypermutation were higher in constructs containing codon-optimized as compared to native S gene sequences, suggesting potential association with increased GC content. These data show that deep sequencing of candidate recombinant vector vaccine constructs in different phases of development is of crucial importance in the development of NDV-based vaccines.

Abstract 341: Study Of ADAR1 Implicates RNA Editing In Vascular Smooth Muscle Cells As A Mechanism Of Coronary Artery Disease Risk

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes. ADAR1 serves a crucial function to edit double stranded RNA (dsRNA) molecules to prevent activation of the dsRNA sensor MDA5 ( IFIH1 ) that stimulates an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism in coronary artery disease (CAD). We hypothesize that smooth muscle cell (SMC) specific RNA editing is required to prevent vascular calcification and inflammation. Here, we demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC). ADAR1 KD in HCASMC causes an ISG response characterized by massive upregulation of key ISG genes (e.g. ISG15, IFI6, IFIH1 ). Importantly, ADAR1 KD stimulates the upregulation of genes with known roles in SMC phenotypic transition and CAD risk, including KLF4 , TCF21 , ATF3 , and ZEB2 . This effect is dependent on activation of MDA5. Additionally, we demonstrate ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro where ADAR1 KD inhibits SMC maturation and dysregulates the calcification gene program in an MDA5 dependent manner. Using scRNAseq data collected from human vascular tissue we show that immunogenic dsRNA are disproportionately expressed in human SMC in atherosclerosis. Similarly, in atherosclerosis prone mice, scRNAseq analysis reveals SMC to have increasing ISG expression as cells transition from mature SMC to a more calcified chondromyocyte phenotype — consistent with MDA5 activation. To further evaluate the role for SMC specific RNA editing in vivo, we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ ). At 2 weeks following SMC- Adar1 KD, mice demonstrate a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. This effect was not seen with an endothelial cell specific deletion model ( Adar1 flox/flox , Cx40 CreERT2 ). Here we provide key evidence to support the observations from human genetics that the ADAR1-dsRNA-MDA5 axis is a causal mechanism in vascular disease and warrants further investigation.

Library Screening Reveals Sequence Motifs That Enable ADAR2 Editing at Recalcitrant Sites.

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in duplex RNA. The inosine product preferentially base pairs with cytidine resulting in an effective A-to-G edit in RNA. ADAR editing can result in a recoding event alongside other alterations to RNA function. A consequence of ADARs' selective activity on duplex RNA is that guide RNAs (gRNAs) can be designed to target an adenosine of interest and promote a desired recoding event. One of ADAR's main limitations is its preference to edit adenosines with specific 5' and 3' nearest neighbor nucleotides (e.g., 5' U, 3' G). Current rational design approaches are well-suited for this ideal sequence context, but limited when applied to difficult-to-edit sites. Here we describe a strategy for the in vitro evaluation of very large libraries of ADAR substrates (En Masse Evaluation of RNA Guides, EMERGe). EMERGe allows for a comprehensive screening of ADAR substrate RNAs that complements current design approaches. We used this approach to identify sequence motifs for gRNAs that enable editing in otherwise difficult-to-edit target sites. A guide RNA bearing one of these sequence motifs enabled the cellular repair of a premature termination codon arising from mutation of the MECP2 gene associated with Rett Syndrome. EMERGe provides an advancement in screening that not only allows for novel gRNA design, but also furthers our understanding of ADARs' specific RNA-protein interactions.

Selective Inhibition of ADAR1 Using 8-Azanebularine-Modified RNA Duplexes.

Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1's role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain's duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5' and 8 bp 3' to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.

Genome-wide profiling of rice Double-stranded RNA-Binding Protein 1-associated RNAs by targeted RNA editing.

RNA-binding proteins (RBPs) play essential roles in regulating gene expression. However, the RNA ligands of RBPs are poorly understood in plants, not least due to the lack of efficient tools for genome-wide identification of RBP-bound RNAs. An RBP-fused adenosine deaminase acting on RNA (ADAR) can edit RBP-bound RNAs, which allows efficient identification of RNA ligands of RBPs in vivo. Here, we report the RNA editing activities of the ADAR deaminase domain (ADARdd) in plants. Protoplast experiments indicated that RBP-ADARdd fusions efficiently edited adenosines within 41 nucleotides (nt) of their binding sites. We then engineered ADARdd to profile the RNA ligands of rice (Oryza sativa) Double-stranded RNA-Binding Protein 1 (OsDRB1). Overexpressing the OsDRB1-ADARdd fusion protein in rice introduced thousands of A-to-G and T-to-C RNA‒DNA variants (RDVs). We developed a stringent bioinformatic approach to identify A-to-I RNA edits from RDVs, which removed 99.7% to 100% of background single-nucleotide variants in RNA-seq data. This pipeline identified a total of 1,798 high-confidence RNA editing (HiCE) sites, which marked 799 transcripts as OsDRB1-binding RNAs, from the leaf and root samples of OsDRB1-ADARdd-overexpressing plants. These HiCE sites were predominantly located in repetitive elements, 3'-UTRs, and introns. Small RNA sequencing also identified 191 A-to-I RNA edits in miRNAs and other sRNAs, confirming that OsDRB1 is involved in sRNA biogenesis or function. Our study presents a valuable tool for genome-wide profiling of RNA ligands of RBPs in plants and provides a global view of OsDRB1-binding RNAs.

Autocatalytic base editing for RNA-responsive translational control

Genetic circuits that control transgene expression in response to pre-defined transcriptional cues would enable the development of smart therapeutics. To this end, here we engineer programmable single-transcript RNA sensors in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert target hybridization into a translational output. Dubbed DART VADAR (Detection and Amplification of RNA Triggers via ADAR), our system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. Amplification is mediated by the expression of a hyperactive, minimal ADAR variant and its recruitment to the edit site via an orthogonal RNA targeting mechanism. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. We leverage DART VADAR to detect single nucleotide polymorphisms and modulate translation in response to endogenous transcript levels in mammalian cells.

The occurrence, characteristics, and adaptation of A-to-I RNA editing in bacteria: A review.

A-to-I RNA editing is a very important post-transcriptional modification or co-transcriptional modification that creates isoforms and increases the diversity of proteins. In this process, adenosine (A) in RNA molecules is hydrolyzed and deaminated into inosine (I). It is well known that ADAR (adenosine deaminase acting on RNA)-dependent A-to-I mRNA editing is widespread in animals. Next, the discovery of A-to-I mRNA editing was mediated by TadA (tRNA-specific adenosine deaminase) in Escherichia coli which is ADAR-independent event. Previously, the editing event S128P on the flagellar structural protein FliC enhanced the bacterial tolerance to oxidative stress in Xoc. In addition, the editing events T408A on the enterobactin iron receptor protein XfeA act as switches by controlling the uptake of Fe3+ in response to the concentration of iron in the environment. Even though bacteria have fewer editing events, the great majority of those that are currently preserved have adaptive benefits. Interestingly, it was found that a TadA-independent A-to-I RNA editing event T408A occurred on xfeA, indicating that there may be other new enzymes that perform a function like TadA. Here, we review recent advances in the characteristics, functions, and adaptations of editing in bacteria.

Core-binding factor fusion downregulation of ADAR2 RNA editing contributes to AML leukemogenesis

AbstractAdenosine-to-inosine RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes, ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than the chronic myeloid leukemia blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core-binding factor (CBF) acute myeloid leukemia (AML) with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO additional exon 9a fusion protein in a dominant-negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of 2 exemplary ADAR2-regulated RNA editing targets coatomer subunit α and component of oligomeric Golgi complex 3 inhibits the clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto, unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

Increasing the Editing Efficiency of the MS2-ADAR System for Site-Directed RNA Editing

Site-directed RNA editing (SDRE) technologies have great potential in gene therapy. Our group has developed a strategy to redirect exogenous adenosine deaminases acting on RNA (ADARs) to specific sites by making editable structures using antisense RNA oligonucleotides. Improving the editing efficiency of the MS2-ADAR system is important in treating undesirable G-to-A point mutations. This work demonstrates an effective strategy to enhance the editing efficiency of this SDRE system. The strategy involves changing the number of MS2 stem-loops on both sides of the antisense RNA and the mismatch base on the antisense part. The enhanced green fluorescent protein (EGFP) with W58X mutation is used as the reporter gene. Subsequently, we adjusted the amount of plasmids for transfection to tune the expression level of the guide RNA, and finally, we observed the fluorescence signal after transfection. After equalizing number of MS2 stem-loops at both sides of the antisense RNA, high editing efficiency was achieved. In the same level of guide RNA expression, when the paired base position was the target uridine, the editing efficiency was higher than cytidine, adenosine, and guanosine.

Characterizing microRNA editing and mutation sites in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder whose pathogenesis is still unclear. MicroRNAs (miRNAs) are a kind of endogenous small non-coding RNAs that play important roles in the post-transcriptional regulation of genes. Recent researches show that miRNAs are edited in multiple ways especially in central nervous systems. A-to-I editing of RNA catalyzed by Adenosine deaminases acting on RNA (ADARs) happens intensively in brain and is also noticed in other organs and tissues. Although miRNAs are widely edited in human brain, miRNA editing in ASD is still largely unexplored. In order to reveal the editing events of miRNAs in ASD, we analyzed 131 miRNA-seq samples from 8 different brain regions of ASD patients and normal controls. We identified 834 editing sites with significant editing levels, of which 70 sites showed significantly different editing levels in the superior frontal gyrus samples of ASD patients (ASD-SFG) when compared with those of control samples. The editing level of an A-to-I editing site in hsa-mir-376a-1 (hsa-mir-376a-1_9_A_g) in ASD-SFG is higher than that of normal controls, and the difference is exaggerated in individuals under 10 years. The increased expression of ADAR1 is consistent with the increased editing level of hsa-mir-376a-1_9_A_g in ASD-SFG samples compared to normal SFG samples. Furthermore, we verify that A-to-I edited hsa-mir-376a-5p directly represses GPR85 and NAPB, which may contribute to the abnormal neuronal development of ASD patients. These results provide new insights into the mechanism of ASD.

The Interplay between RNA Editing Regulator ADAR1 and Immune Environment in Colorectal Cancer.

An abnormality in the regulation of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyzed adenosine-to-inosine (A-to-I) RNA editing, was closely associated with the highly aggressive biologic behavior and poor prognosis in many malignancies. In the present study, we aimed to investigate the relationship among transcript factors-microRNAs regulatory network, immune environment, and ADAR gene in colorectal carcinoma (CRC). The association among the expression levels of ADAR mRNA and copy number variation, methylation, and mutation status were comprehensively analyzed using cBioPortal, Wanderer, and UALCAN databases in CRC datasets. ADAR-transcript factors (TFs) and ADAR-miRNA regulation networks were constructed by Cistrome Cancer and miRWalk2.0, respectively. The full network and subnetworks for ADAR coexpression genes were constructed using the STRING database and visualized by the MCODE module of the Cytoscape app. The relationship between ADAR mRNA expression and the abundance of infiltrating immune cells in CRC patients was explored by the Tumor Immune Estimation Resource, CIBERSORT, and single-gene gene set enrichment analysis (GSEA). ADAR mRNA was elevated and was a cancer essential gene in CRC. ADAR mRNA and transcripts P110 were significantly elevated in CRC compared to normal controls. Low-level methylation in the promoter region and high copy number amplification of ADAR were responsible for high levels of ADAR mRNA expression. ADAR coexpression genes were mainly involved in immunoregulation, especially T-lymphocyte activation. Hub genes, including CD2, CD274, and FASLG, were also significantly upregulated in the ADAR-high group compared to the control group. Besides, M1 macrophages were enriched in the ADAR-high group compared to the control group. This study demonstrated that ADAR, a new essential gene, was involved in the immune regulator and was a novel immune treatment target in CRC.

Elevated ADAR expression is significantly linked to shorter overall survival and immune infiltration in patients with lung adenocarcinoma.

To date, few studies have investigated whether the RNA-editing enzymes adenosine deaminases acting on RNA (ADARs) influence RNA functioning in lung adenocarcinoma (LUAD). To investigate the role of ADAR in lung cancer, we leveraged the advantages of The Cancer Genome Atlas (TCGA) database, from which we obtained transcriptome data and clinical information from 539 patients with LUAD. First, we compared ARAR expression levels in LUAD tissues with those in normal lung tissues using paired and unpaired analyses. Next, we evaluated the influence of ADARs on multiple prognostic indicators, including overall survival at 1, 3 and 5 years, as well as disease-specific survival and progression-free interval, in patients with LUAD. We also used Kaplan-Meier survival curves to estimate overall survival and Cox regression analysis to assess covariates associated with prognosis. A nomogram was constructed to validate the impact of the ADARs and clinicopathological factors on patient survival probabilities. The volcano plot and heat map revealed the differentially expressed genes associated with ADARs in LUAD. Finally, we examined ADAR expression versus immune cell infiltration in LUAD using Spearman's analysis. Using the Gene Expression Profiling Interactive Analysis (GEPIA2) database, we identified the top 100 genes most significantly correlated with ADAR expression, constructed a protein-protein interaction network and performed a Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analysis on these genes. Our results demonstrate that ADARs are overexpressed in LUAD and correlated with poor patient prognosis. ADARs markedly increase the infiltration of T central memory, T helper 2 and T helper cells, while reducing the infiltration of immature dendritic, dendritic and mast cells. Most immune response markers, including T cells, tumor-associated macrophages, T cell exhaustion, mast cells, macrophages, monocytes and dendritic cells, are closely correlated with ADAR expression in LUAD.

Mutational Patterns Observed in SARS-CoV-2 Genomes Sampled From Successive Epochs Delimited by Major Public Health Events in Ontario, Canada: Genomic Surveillance Study

Background The emergence of SARS-CoV-2 variants with mutations associated with increased transmissibility and virulence is a public health concern in Ontario, Canada. Characterizing how the mutational patterns of the SARS-CoV-2 genome have changed over time can shed light on the driving factors, including selection for increased fitness and host immune response, that may contribute to the emergence of novel variants. Moreover, the study of SARS-CoV-2 in the microcosm of Ontario, Canada can reveal how different province-specific public health policies over time may be associated with observed mutational patterns as a model system. Objective This study aimed to perform a comprehensive analysis of single base substitution (SBS) types, counts, and genomic locations observed in SARS-CoV-2 genomic sequences sampled in Ontario, Canada. Comparisons of mutational patterns were conducted between sequences sampled during 4 different epochs delimited by major public health events to track the evolution of the SARS-CoV-2 mutational landscape over 2 years. Methods In total, 24,244 SARS-CoV-2 genomic sequences and associated metadata sampled in Ontario, Canada from January 1, 2020, to December 31, 2021, were retrieved from the Global Initiative on Sharing All Influenza Data database. Sequences were assigned to 4 epochs delimited by major public health events based on the sampling date. SBSs from each SARS-CoV-2 sequence were identified relative to the MN996528.1 reference genome. Catalogues of SBS types and counts were generated to estimate the impact of selection in each open reading frame, and identify mutation clusters. The estimation of mutational fitness over time was performed using the Augur pipeline. Results The biases in SBS types and proportions observed support previous reports of host antiviral defense activity involving the SARS-CoV-2 genome. There was an increase in U>C substitutions associated with adenosine deaminase acting on RNA (ADAR) activity uniquely observed during Epoch 4. The burden of novel SBSs observed in SARS-CoV-2 genomic sequences was the greatest in Epoch 2 (median 5), followed by Epoch 3 (median 4). Clusters of SBSs were observed in the spike protein open reading frame, ORF1a, and ORF3a. The high proportion of nonsynonymous SBSs and increasing dN/dS metric (ratio of nonsynonymous to synonymous mutations in a given open reading frame) to above 1 in Epoch 4 indicate positive selection of the spike protein open reading frame. Conclusions Quantitative analysis of the mutational patterns of the SARS-CoV-2 genome in the microcosm of Ontario, Canada within early consecutive epochs of the pandemic tracked the mutational dynamics in the context of public health events that instigate significant shifts in selection and mutagenesis. Continued genomic surveillance of emergent variants will be useful for the design of public health policies in response to the evolving COVID-19 pandemic.

Characterization of SARS-CoV-2 Mutational Signatures from 1.5+ Million Raw Sequencing Samples.

We present a large-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) substitutions, considering 1,585,456 high-quality raw sequencing samples, aimed at investigating the existence and quantifying the effect of mutational processes causing mutations in SARS-CoV-2 genomes when interacting with the human host. As a result, we confirmed the presence of three well-differentiated mutational processes likely ruled by reactive oxygen species (ROS), apolipoprotein B editing complex (APOBEC), and adenosine deaminase acting on RNA (ADAR). We then evaluated the activity of these mutational processes in different continental groups, showing that some samples from Africa present a significantly higher number of substitutions, most likely due to higher APOBEC activity. We finally analyzed the activity of mutational processes across different SARS-CoV-2 variants, and we found a significantly lower number of mutations attributable to APOBEC activity in samples assigned to the Omicron variant.

Filamin A pre-mRNA editing modulates vascularization and tumor growth

Adenosine to inosine (A to I) editing is mediated by adenosine deaminases acting on RNA (ADAR) enzymes. Inosines are interpreted as guanosines by the translational machinery. Consequently, A to I editing in mRNAs can lead to their recoding and the formation of proteins not encoded in the genome. Filamin A is an actin-crosslinking protein. A to I editing in the filamin pre-mRNA leads to the exchange of a glutamine to an arginine in a highly interactive domain of the protein. However, the consequences of this editing event are still poorly understood. Here we show, using transgenic mice expressing either constitutively edited or constitutively uneditable filamin A that filamin A editing critically controls angiogenesis in tumors but also in a mouse ischemia model. Hyper-editing reduces angiogenesis, while hypoediting leads to increased angiogenesis, possibly byaltering vascular endothelial growth factor receptor 2 (VEGFR2) turnover. Further, FLNA editing of the tumor itself seemingly affects its metastatic potential by changing its interaction with the extracellular matrix. We therefore identify filamin A editing as a critical component for angiogenesis, tumor growth, and metastasis formation.

The hepatitis delta virus and chronic hepatitis D.

The hepatitis delta virus and chronic hepatitis D.