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Background Adenosine deaminase (ADA) and osteopontin (OPN) may play opposing roles in the pathogenesis of COPD. Deficiency of ADA results in enhanced adenosine signaling which up-regulates OPN expression. Although statins suppress OPN in cancer cells, little is known about their effects on ADA and OPN in COPD patients. Methods We extended a previous randomized double-blind placebo crossover study to investigate the effects of simvastatin (20 mg/day) on sputum ADA and OPN expression and explored the underlying signaling pathways involved by conducting in vitro experiments with cigarette smoke extract (CSE)-treated monocyte-derived macrophages (MDM) from COPD patients and healthy subjects. Results Simvastatin decreased sputum IL-13, OPN and CD73, while increasing ADA expression, irrespective of inhaled corticosteroid treatment and smoking status in parallel to increased inosine levels. The degree of simvastatin-restored ADA activity was significantly correlated with the magnitude of changes in pre-bronchodilator FEV 1 . Mechanistic exploration showed that CSE enhanced the expression of IL-13, which induced an increase in OPN and inhibited ADA mRNA accumulation in MDM from COPD patients but not healthy subjects through a STAT6-dependent mechanism. Simvastatin treatment inhibited IL-13 transcription in a dose-dependent manner, and therefore diminished the IL-13-induced increase in OPN and restored IL-13-suppressed ADA. There was no effect of simvastatin on adenosine receptors in CSE-stimulated MDM, indicating that its effects were on the adenosine pathway. Conclusion Simvastatin reversed IL-13-suppressed ADA activity that leads to the down-regulation of adenosine signaling and therefore inhibits OPN expression through the direct inhibition of IL-13-activated STAT6 pathway. Inhibition of IL-13 may reverse the imbalance between ADA and OPN in COPD and therefore may prevent COPD progression.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA) 1 . In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi–Goutières syndrome (AGS) 2 . In mice, complete loss of ADAR1 activity is embryonically lethal 3 – 6 , and mutations similar to those found in patients with AGS cause autoinflammation 7 – 12 . Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR 3 , 7 – 10 , 13 , 14 . Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar -knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function. In addition to its role in suppressing MDA5 and PKR activation, ADAR1 is a negative regulator of ZPB1-mediated apoptosis and necroptosis, providing insights into the pathology of Aicardi–Goutières syndrome.

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.

In the absence of DHX9, circular RNAs accumulate and transcription and translation are dysregulated—effects that are exacerbated by concomitant depletion of the RNA-editing enzyme ADAR. DHX9 suppresses Alu-derived defects In the human genome, there are more than a million copies of the Alu transposable element. Movement of Alu elements is a common source of mutations, but as insertions usually occur in non-coding regions, they are often without discernible effect. Alu elements located near one another in an inverted orientation will form secondary structures that may affect various nuclear processes. Asifa Akhtar and colleagues find that the RNA helicase, DHX9, binds transcribed ‘IRAlus’ (inverted repeat Alu elements). In the absence of DHX9, circular RNAs accumulate, and transcription and translation are dysregulated. These effects are further exacerbated by co-depletion of DHX9 and ADAR p150, an interferon-inducible RNA modification enzyme. The authors conclude that these proteins protect against transposon insertion, which can have deleterious effects on gene expression. Transposable elements are viewed as ‘selfish genetic elements’, yet they contribute to gene regulation and genome evolution in diverse ways 1 . More than half of the human genome consists of transposable elements 2 . Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome 2 . Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability 3 . Alu elements are the main targets of the RNA-editing enzyme ADAR 4 and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC 5 , but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant 6 nuclear RNA helicase 7 , binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated ( P  = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not ( P  = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated ( P  = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated ( P  = 0.017) and controls ( P  = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.

Key Points A-to-I RNA editing is catalysed by adenosine deaminases acting on RNA (ADARs). Three mammalian ADAR genes ( ADAR1 , ADAR2 and ADAR3 ) with common functional domains have been identified. Protein-coding sequences of a limited number of genes, such as glutamate receptor GRIA2 and serotonin receptor HTR2C , are edited, resulting in dramatic alterations of protein functions. Deficiencies in A-to-I RNA editing cause human diseases and pathophysiology. Genome-wide screening has identified numerous A-to-I editing sites in inverted Alu repeats located in non-coding regions of mRNAs. Alu editing in these transcripts is likely to affect many cellular processes. The biogenesis and function of certain miRNAs is regulated by editing of the primary miRNAs (pri-miRNAs). ADAR1 forms a complex with Dicer to promote the efficacy of miRNA processing and RNA interference (RNAi) in developing embryos. ADAR enzymes convert adenosine to inosine (A-to-I editing) at numerous double-stranded Alu repeats in human transcripts, thereby affecting many cellular processes. Primary microRNAs (miRNAs) are also edited, and ADAR1 directly interacts with Dicer, resulting in the modulation of miRNA expression and activity and of downstream gene expression programmes during embryogenesis. Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR–Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage. A new DNA ‘base editor’ can change targeted A•T base pairs to G•C, allowing disease-associated mutations to be corrected and disease-suppressing mutations to be introduced into cells. Base editing steps forward In 2016, David Liu and colleagues developed a DNA 'base editor'—a system that would make it possible to change C•G base pairs to T•A base pairs within DNA without introducing double-stranded breaks. This approach involves tethering of a cytidine deaminase to an inactive RNA-guided Cas9 complex that enables site selectivity. However, this system was unable to correct about half of the single nucleotide polymorphisms that are known to be pathogenic. Now, David Liu and collaborators describe the next step in genomic base editing technology, designed to tackle the conversion of A•T base pairs to G•C base pairs. Beginning with a bacterial adenosine deaminase that acts on RNA, they used seven rounds of selection and refinement to produce ABE7.10. This enzyme, again tethered to an inactive RNA-guided Cas9 complex, uses DNA as a substrate and resulted in an average correction efficiency of 53% across multiple sites and contexts in the genome, with a very low mutagenic background. Importantly, the system can be used both to correct disease-associated single nucleotide polymorphisms and to introduce disease-suppressing ones.

One of the most prevalent forms of post-transcritpional RNA modification is the conversion of adenosine nucleosides to inosine (A-to-I), mediated by the ADAR family of enzymes. The functional requirement and regulatory landscape for the majority of A-to-I editing events are, at present, uncertain. Recent studies have identified key in vivo functions of ADAR enzymes, informing our understanding of the biological importance of A-to-I editing. Large-scale studies have revealed how editing is regulated both in cis and in trans . This review will explore these recent studies and how they broaden our understanding of the functions and regulation of ADAR-mediated RNA editing.

Adenosine deaminase 2 (ADA2) is a protein with at least two functions. It is a growth factor affecting leukocytes and endothelial cells and an enzyme that influences purine metabolism. This study shows that mutant ADA2 causes polyarteritis nodosa. Polyarteritis nodosa, first described in 1866, 1 is a systemic necrotizing vasculitis that affects medium and small muscular arteries. 2 , 3 The ensuing tissue ischemia can affect any organ, including the skin, musculoskeletal system, kidneys, gastrointestinal tract, and the cardiovascular and nervous systems. Polyarteritis nodosa is usually diagnosed in middle age or later but can appear in childhood. 2 , 4 , 5 The diagnosis remains challenging despite classification criteria for adults 6 and children, 7 because polyarteritis nodosa frequently presents with nonspecific constitutional symptoms, and organ involvement and disease severity are highly varied. Polyarteritis nodosa is most often primary, but in adults it may be associated . . .

Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1E861A, where E861A denotes Glu861→Ala861). Adar1E861A/E861A embryos died at ∼E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)–sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3′ untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1E861A/E861A were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.