HSV-1 miRNAs are post-transcriptionally edited in latently infected human ganglia.

IMPORTANCE: Herpes simplex virus 1 is an important human pathogen that has been intensively studied for many decades. Nevertheless, the molecular mechanisms regulating its establishment, maintenance, and reactivation from latency are poorly understood. Here, we show that HSV-1-encoded miR-H2 is post-transcriptionally edited in latently infected human tissues. Hyperediting of viral miRNAs increases the targeting potential of these miRNAs and may play an important role in regulating latency. We show that the edited miR-H2 can target ICP4, an essential viral protein. Interestingly, we found no evidence of hyperediting of its homolog, miR-H2, which is expressed by the closely related virus HSV-2. The discovery of post-translational modifications of viral miRNA in the latency phase suggests that these processes may also be important for other non-coding viral RNA in the latency phase, including the intron LAT, which in turn may be crucial for understanding the biology of this virus.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors.

PURPOSE: Patient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs). METHODS: 94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively. RESULTS: Exome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S - adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways. CONCLUSION: The availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Contribution of A-to-I RNA editing, M6A RNA Methylation, and Alternative Splicing to physiological brain aging and neurodegenerative diseases.

Aging is a physiological and progressive phenomenon in all organisms' life cycle, characterized by the accumulation of degenerative processes triggered by several alterations within molecular pathways. These changes compromise cell fate, resulting in the loss of functions in tissues throughout the body, including the brain. Physiological brain aging has been linked to structural and functional alterations, as well as to an increased risk of neurodegenerative diseases. Post-transcriptional RNA modifications modulate mRNA coding properties, stability, translatability, expanding the coding capacity of the genome, and are involved in all cellular processes. Among mRNA post-transcriptional modifications, the A-to-I RNA editing, m6A RNA Methylation and Alternative Splicing play a critical role in all the phases of a neuronal cell life cycle and alterations in their mechanisms of action significantly contribute to aging and neurodegeneration. Here we review our current understanding of the contribution of A-to-I RNA editing, m6A RNA Methylation, and Alternative Splicing to physiological brain aging process and neurodegenerative diseases.

Vaccines and more: The response of Dark Web marketplaces to the ongoing COVID-19 pandemic.

Early analyses revealed that dark web marketplaces (DWMs) started offering COVID-19 related products (e.g., masks and COVID-19 tests) as soon as the COVID-19 pandemic started, when these goods were in shortage in the traditional economy. Here, we broaden the scope and depth of previous investigations by considering how DWMs responded to an ongoing pandemic after the initial shock. Our dataset contains listings from 194 DWMs collected until July 2021. We start by focusing on vaccines. We find 248 listings offering approved vaccines, like Pfizer/BioNTech and AstraZeneca, as well as vendors offering fabricated proofs of vaccination and COVID-19 passports. Then, we consider COVID-19 related products. We show that, as the regular economy has become able to satisfy the demand of these goods, DWMs have decreased their offer. Next, we analyse the profile of vendors of COVID-19 related products and vaccines. We find that most of them are specialized in a single type of listings and are willing to ship worldwide. Finally, we consider a broader set of listings mentioning COVID-19, in order to assess the general impact of the pandemic on the broader activity of DWMs. Among 10,330 such listings, we show that recreational drugs are the most affected among traditional DWMs product, with COVID-19 mentions steadily increasing since March 2020. We anticipate that our results will be of interest to researchers, practitioners, and law enforcement agencies focused on the study and safeguard of public health.

ADAR2 Protein Is Associated with Overall Survival in GBM Patients and Its Decrease Triggers the Anchorage-Independent Cell Growth Signature.

BACKGROUND: Epitranscriptomic mechanisms, such as A-to-I RNA editing mediated by ADAR deaminases, contribute to cancer heterogeneity and patients' stratification. ADAR enzymes can change the sequence, structure, and expression of several RNAs, affecting cancer cell behavior. In glioblastoma, an overall decrease in ADAR2 RNA level/activity has been reported. However, no data on ADAR2 protein levels in GBM patient tissues are available; and most data are based on ADARs overexpression experiments. METHODS: We performed IHC analysis on GBM tissues and correlated ADAR2 levels and patients' overall survival. We silenced ADAR2 in GBM cells, studied cell behavior, and performed a gene expression/editing analysis. RESULTS: GBM tissues do not all show a low/no ADAR2 level, as expected by previous studies. Although, different amounts of ADAR2 protein were observed in different patients, with a low level correlating with a poor patient outcome. Indeed, reducing the endogenous ADAR2 protein in GBM cells promotes cell proliferation and migration and changes the cell's program to an anchorage-independent growth mode. In addition, deep-seq data and bioinformatics analysis indicated multiple RNAs are differently expressed/edited upon siADAR2. CONCLUSION: ADAR2 protein is an important deaminase in GBM and its amount correlates with patient prognosis.

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

ADAR1 is a new target of METTL3 and plays a pro-oncogenic role in glioblastoma by an editing-independent mechanism.

BACKGROUND: N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) RNA editing are two of the most abundant RNA modification events affecting adenosines in mammals. Both these RNA modifications determine mRNA fate and play a pivotal role in tumor development and progression. RESULTS: Here, we show that METTL3, upregulated in glioblastoma, methylates ADAR1 mRNA and increases its protein level leading to a pro-tumorigenic mechanism connecting METTL3, YTHDF1, and ADAR1. We show that ADAR1 plays a cancer-promoting role independently of its deaminase activity by binding CDK2 mRNA, underlining the importance of ADARs as essential RNA-binding proteins for cell homeostasis as well as cancer progression. Additionally, we show that ADAR1 knockdown is sufficient to strongly inhibit glioblastoma growth in vivo. CONCLUSIONS: Hence, our findings underscore METTL3/ADAR1 axis as a novel crucial pathway in cancer progression that connects m6A and A-to-I editing post-transcriptional events.

Dark Web Marketplaces and COVID-19: before the vaccine.

The COVID-19 pandemic has reshaped the demand for goods and services worldwide. The combination of a public health emergency, economic distress, and misinformation-driven panic have pushed customers and vendors towards the shadow economy. In particular, dark web marketplaces (DWMs), commercial websites accessible via free software, have gained significant popularity. Here, we analyse 851,199 listings extracted from 30 DWMs between January 1, 2020 and November 16, 2020. We identify 788 listings directly related to COVID-19 products and monitor the temporal evolution of product categories including Personal Protective Equipment (PPE), medicines (e.g., hydroxyclorochine), and medical frauds. Finally, we compare trends in their temporal evolution with variations in public attention, as measured by Twitter posts and Wikipedia page visits. We reveal how the online shadow economy has evolved during the COVID-19 pandemic and highlight the importance of a continuous monitoring of DWMs, especially now that real vaccines are available and in short supply. We anticipate our analysis will be of interest both to researchers and public agencies focused on the protection of public health.

MicroRNA Editing Detection and Function: A Combined In Silico and Experimental Approach for the Identification and Validation of Putative Oncogenic Targets.

MicroRNAs (miRNAs) are a class of ~22 nt noncoding RNAs playing essential roles in the post-transcriptional regulation of gene expression, cell proliferation, and cell differentiation and are often found deregulated in several diseases including cancer.The A-to-I RNA editing, mediated by ADAR enzymes, is a diffuse post-transcriptional mechanism that converts the genetically coded adenosine (A) into inosine (I) at the RNA level. Among different RNA targets, the ADAR enzymes can also edit miRNA precursors. Specifically, a single nucleotide change (A/I) lying within the mature miRNA can alter the miRNA binding specificity and redirect the edited miRNA to a different mRNA target. In several cancer types a consistent deregulation of A-to-I RNA editing machinery also involves important miRNAs (either oncomiRs or tumor-suppressor miRNAs). Herein we describe a combined in silico and experimental approach for the detection of edited miRNAs and the identification and validation of their target genes potentially involved in cancer progression or invasion.

From code to market: Network of developers and correlated returns of cryptocurrencies.

"Code is law" is the founding principle of cryptocurrencies. The security, transferability, availability, and other properties of crypto-assets are determined by the code through which they are created. If code is open source, as is customary for cryptocurrencies, this would prevent manipulations and grant transparency to users and traders. However, this approach considers cryptocurrencies as isolated entities, neglecting possible connections between them. Here, we show that 4% of developers contribute to the code of more than one cryptocurrency and that the market reflects these cross-asset dependencies. In particular, we reveal that the first coding event linking two cryptocurrencies through a common developer leads to the synchronization of their returns. Our results identify a clear link between the collaborative development of cryptocurrencies and their market behavior. More broadly, they reveal a so-far overlooked systemic dimension for the transparency of code-based ecosystems that will be of interest for researchers, investors, and regulators.

De Novo A-to-I RNA Editing Discovery in lncRNA.

BACKGROUND: Adenosine to inosine (A-to-I) RNA editing is the most frequent editing event in humans. It converts adenosine to inosine in double-stranded RNA regions (in coding and non-coding RNAs) through the action of the adenosine deaminase acting on RNA (ADAR) enzymes. Long non-coding RNAs, particularly abundant in the brain, account for a large fraction of the human transcriptome, and their important regulatory role is becoming progressively evident in both normal and transformed cells. RESULTS: Herein, we present a bioinformatic analysis to generate a comprehensive inosinome picture in long non-coding RNAs (lncRNAs), using an ad hoc index and searching for de novo editing events in the normal brain cortex as well as in glioblastoma, a highly aggressive human brain cancer. We discovered >10,000 new sites and 335 novel lncRNAs that undergo editing, never reported before. We found a generalized downregulation of editing at multiple lncRNA sites in glioblastoma samples when compared to the normal brain cortex. CONCLUSION: Overall, our study discloses a novel layer of complexity that controls lncRNAs in the brain and brain cancer.

Aptamer-Based In Vivo Therapeutic Targeting of Glioblastoma.

Glioblastoma (GBM) is the most aggressive, infiltrative, and lethal brain tumor in humans. Despite the extensive advancement in the knowledge about tumor progression and treatment over the last few years, the prognosis of GBM is still very poor due to the difficulty of targeting drugs or anticancer molecules to GBM cells. The major challenge in improving GBM treatment implicates the development of a targeted drug delivery system, capable of crossing the blood-brain barrier (BBB) and specifically targeting GBM cells. Aptamers possess many characteristics that make them ideal novel therapeutic agents for the treatment of GBM. They are short single-stranded nucleic acids (RNA or ssDNA) able to bind to a molecular target with high affinity and specificity. Several GBM-targeting aptamers have been developed for imaging, tumor cell isolation from biopsies, and drug/anticancer molecule delivery to the tumor cells. Due to their properties (low immunogenicity, long stability, and toxicity), a large number of aptamers have been selected against GBM biomarkers and tested in GBM cell lines, while only a few of them have also been tested in in vivo models of GBM. Herein, we specifically focus on aptamers tested in GBM in vivo models that can be considered as new diagnostic and/or therapeutic tools for GBM patients' treatment.

Quantifying RNA Editing in Deep Transcriptome Datasets.

Massive transcriptome sequencing through the RNAseq technology has enabled quantitative transcriptome-wide investigation of co-/post-transcriptional mechanisms such as alternative splicing and RNA editing. The latter is abundant in human transcriptomes in which million adenosines are deaminated into inosines by the ADAR enzymes. RNA editing modulates the innate immune response and its deregulation has been associated with different human diseases including autoimmune and inflammatory pathologies, neurodegenerative and psychiatric disorders, and tumors. Accurate profiling of RNA editing using deep transcriptome data is still a challenge, and the results depend strongly on processing and alignment steps taken. Accurate calling of the inosinome repertoire, however, is required to reliably quantify RNA editing and, in turn, investigate its biological and functional role across multiple samples. Using real RNAseq data, we demonstrate the impact of different bioinformatics steps on RNA editing detection and describe the main metrics to quantify its level of activity.

The Interplay between CD27dull and CD27bright B Cells Ensures the Flexibility, Stability, and Resilience of Human B Cell Memory.

Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27dull and CD27bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27dull and CD27bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27dull into the CD27bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27dull MBCs transit to the more differentiated CD27bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27dull MBCs that expand and differentiate in response to change.

AKT-Dependent Phosphorylation of ADAR1p110 and ADAR2 Represents a New and Important Link Between Cell Signaling and RNA Editing.

RNA editing is a process by which nascent RNA transcripts are covalently modified, thus enhancing the complexity of the transcriptome. The most common modifications are deaminations of adenosine to inosine at sites of complex RNA secondary structure, a process that is carried out by the adenosine deaminase acting on double-strand RNA (ADAR) family of RNA editases. Although much has been learned about the ADAR family members since their discovery, very little information on their post-transcriptional regulation has been reported. Similar to most proteins, the ADAR family members are post-translationally modified at multiple sites. We recently reported that members of the AKT kinase family directly phosphorylate ADAR1p110 and ADAR2 on a conserved threonine within the catalytic domain of the protein. Phosphorylation was observed to differentially inhibit the enzymatic activity of the ADAR proteins toward known RNA substrates. The direct downstream involvement of the AKT kinases in multiple major signaling pathways associated with cell survival, growth, glucose metabolism (insulin signaling), and differentiation is well established; thus, the AKT kinases represent a link between ADAR-dependent A-to-I editing and major signal transduction pathways that are necessary for cell maintenance and development.

Increased adenosine-to-inosine RNA editing in rheumatoid arthritis.

OBJECTIVE: Adenosine-to-inosine (A-to-I) RNA editing of Alu retroelements is a primate-specific mechanism mediated by adenosine deaminases acting on RNA (ADARs) that diversifies transcriptome by changing selected nucleotides in RNA molecules. We tested the hypothesis that A-to-I RNA editing is altered in rheumatoid arthritis (RA). METHODS: Synovium expression analysis of ADAR1 was investigated in 152 RA patients and 50 controls. Peripheral blood mononuclear cells derived from 14 healthy subjects and 19 patients with active RA at baseline and after 12-week treatment were examined for ADAR1p150 and ADAR1p110 isoform expression by RT-qPCR. RNA editing activity was analysed by AluSx+ Sanger-sequencing of cathepsin S, an extracellular matrix degradation enzyme involved in antigen presentation. RESULTS: ADAR1 was significantly over-expressed in RA synovium regardless of disease duration. Similarly, ADAR1p150 isoform expression was significantly increased in the blood of active RA patients. Individual nucleotide analysis revealed that A-to-I RNA editing rate was also significantly increased in RA patients. Both baseline ADAR1p150 expression and individual adenosine RNA editing rate of cathepsin S AluSx+ decreased after treatment only in those patients with good clinical response. Upregulation of the expression and/or activity of the RNA editing machinery were associated with a higher expression of edited Alu-enriched genes including cathepsin S and TNF receptor-associated factors 1,2,3 and 5. CONCLUSION: A previously unrecognized regulation and role of ADAR1p150-mediated A-to-I RNA editing in post-transcriptional control in RA underpins therapeutic response and fuels inflammatory gene expression, thus representing an interesting therapeutic target.

Signal Transduction in Ribosome Biogenesis: A Recipe to Avoid Disaster.

Energetically speaking, ribosome biogenesis is by far the most costly process of the cell and, therefore, must be highly regulated in order to avoid unnecessary energy expenditure. Not only must ribosomal RNA (rRNA) synthesis, ribosomal protein (RP) transcription, translation, and nuclear import, as well as ribosome assembly, be tightly controlled, these events must be coordinated with other cellular events, such as cell division and differentiation. In addition, ribosome biogenesis must respond rapidly to environmental cues mediated by internal and cell surface receptors, or stress (oxidative stress, DNA damage, amino acid depletion, etc.). This review examines some of the well-studied pathways known to control ribosome biogenesis (PI3K-AKT-mTOR, RB-p53, MYC) and how they may interact with some of the less well studied pathways (eIF2α kinase and RNA editing/splicing) in higher eukaryotes to regulate ribosome biogenesis, assembly, and protein translation in a dynamic manner.

AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.

Murine thymoma viral oncogene homolog (AKT) kinases target both cytosolic and nuclear substrates for phosphorylation. Whereas the cytosolic substrates are known to be closely associated with the regulation of apoptosis and autophagy or metabolism and protein synthesis, the nuclear substrates are, for the most part, poorly understood. To better define the role of nuclear AKT, potential AKT substrates were isolated from the nuclear lysates of leukemic cell lines using a phosphorylated AKT substrate antibody and identified in tandem mass spectrometry. Among the proteins identified was adenosine deaminase acting on RNA (ADAR)1p110, the predominant nuclear isoform of the adenosine deaminase acting on double-stranded RNA. Coimmunoprecipitation studies and in vitro kinase assays revealed that AKT-1, -2, and -3 interact with both ADAR1p110 and ADAR2 and phosphorylate these RNA editases. Using site-directed mutagenesis of suspected AKT phosphorylation sites, AKT was found to primarily phosphorylate ADAR1p110 and ADAR2 on T738 and T553, respectively, and overexpression of the phosphomimic mutants ADAR1p110 (T738D) and ADAR2 (T553D) resulted in a 50-100% reduction in editase activity. Thus, activation of AKT has a direct and major impact on RNA editing.-Bavelloni, A., Focaccia, E., Piazzi, M., Raffini, M., Cesarini, V., Tomaselli, S., Orsini, A., Ratti, S., Faenza, I., Cocco, L., Gallo, A., Blalock, W. L. AKT-dependent phosphorylation of the adenosine deaminases ADAR-1 and -2 inhibits deaminase activity.