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Introduction: Fibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection., Methods: The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene -based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression., Results: WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor β1 (TGF-β1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-β1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts., Conclusion: These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions., Competing Interests: Competing interests: RCC received grants from GSK via her institution during the conduct of the study; consultancy fees from Pieris Pharmaceuticals, consultancy fees from Chiesi, consultancy fees from Theravance Biopharma outside the submitted work; and RCC spouse was an employee of GSK during the time this work was conducted. This potential COI was managed by a research framework agreement between UCL and GSK. DG and PFM received salary from this research framework agreement. RM, AT, LME and AB were employees of GlaxoSmithKline during the time this work was performed and hold GSK shares. AJF has received a research grant from GSK to support work contained in this manuscript. RH and GM were employees of Epistem with a commercial interest in the provision of services in the area of investigation. TMM has, via his institution, received industry-academic funding from GSK, R&D and UCB and has received consultancy or speakers fees from Apellis, AstraZeneca, Bayer, Blade Therapeutics, Boehringer Ingelheim, Bristol-Myers Squibb, Galapagos, GlaxoSmithKline R&D, Indalo, Novartis, Pliant, ProMetic, Respivnat, Roche, Samumed and UCB. MJH reports non-financial support from Achilles Therapeutics outside the submitted work. MP, RJM and REH have nothing to disclose., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Molecules capable of directing changes to nucleic acid sequences are powerful tools for molecular biology and promising candidates for the therapeutic correction of disease-causing mutations. However, unwanted reactions at off-target sites complicate their use. Here we report selective combinations of mutant editing enzyme and directing oligonucleotide. Mutations in human ADAR2 (adenosine deaminase acting on RNA 2) that introduce aromatic amino acids at position 488 reduce background RNA editing. This residue is juxtaposed to the nucleobase that pairs with the editing site adenine, suggesting a steric clash for the bulky mutants. Replacing this nucleobase with a hydrogen atom removes the clash and restores editing activity. A crystal structure of the E488Y mutant bound to abasic site-containing RNA shows the accommodation of the tyrosine side chain. Finally, we demonstrate directed RNA editing in vitro and in human cells using mutant ADAR2 proteins and modified guide RNAs with reduced off-target activity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Adenosine (A) to inosine (I) RNA editing is important for life in metazoan organisms. Dysregulation or mutations that compromise the efficacy of A to I editing results in neurological disorders and a shorten life span. These reactions are catalyzed by adenosine deaminases acting on RNA (ADARs), which hydrolytically deaminate adenosines in regions of duplex RNA. Because inosine mimics guanosine in hydrogen bonding, this prolific RNA editing alters the sequence and structural information in the RNA landscape. Aicardi-Goutières syndrome (AGS) is a severe childhood autoimmune disease that is one of a broader set of inherited disorders characterized by constitutive upregulation of type I interferon (IFN) referred to as type I interferonopathies. AGS is caused by mutations in multiple genes whose protein products, including ADAR1, are all involved in nucleic acid metabolism or sensing. The recent crystal structures of human ADAR2 deaminase domain complexed with duplex RNA substrates enabled modeling of how AGS causing mutations may influence RNA binding and catalysis. The mutations can be broadly characterized into three groups; mutations on RNA-binding loops that directly affect RNA binding, "second-layer" mutations that can alter the disposition of RNA-binding loops, and mutations that can alter the position of an α-helix bearing an essential catalytic residue.

Background: CD248 or Endosialin is a transmembrane molecule expressed in stromal cells binding to extracellular matrix (ECM) components. It has been previously implicated in kidney fibrosis, rheumatoid arthritis as well as in tumour-stromal interactions. This study investigates the role of CD248 in the pathogenesis of fibrotic diseases in Idiopathic Pulmonary Fibrosis (IPF)., Methods: CD248 quantitative immunohistochemistry (IHC) was performed on lung samples from 22 IPF patients and its expression was assayed in cultured pulmonary fibroblasts and epithelial cells. Effects of CD248 silencing was evaluated on fibroblast proliferation and myofibroblast differentiation., Results: IHC revealed strong CD248 expression in mesenchymal cells of normal lung structures such as pleura and adventitia but not in epithelium. Fibrotic areas showed markedly stronger staining than unaffected lung tissue. The extent of CD248 staining showed a significant negative correlation to lung function parameters FEV1, FVC, TLC, and TLCO (r2 > 0 · 35, p < 0 · 01). CD248 protein levels were significantly greater in IPF-derived lung fibroblasts vs normal lung fibroblasts (p < 0 · 01) and CD248 silencing significantly reduced the proliferation of lung fibroblasts, but did not affected myofibroblast differentiation., Conclusion: We conclude that CD248 overexpression is possibly involved in the pathogenesis of IPF and it has potential as a disease severity marker. Given that CD248 ligands are collagen type I, IV and fibronectin, we hypothesise that CD248 signalling represents a novel matrix-fibroblast interaction that may be a potential therapeutic target in IPF.

Ribonucleoside analogues bearing terminal alkynes, including 7-ethynyl-8-aza-7-deazaadenosine (7-EAA), are useful for RNA modification applications. However, although alkyne- and triazole-bearing ribonucleosides are in widespread use, very little information is available on the impact of these modifications on RNA structure. By solving crystal structures for RNA duplexes containing these analogues, we show that, like adenosine, 7-EAA and a triazole derived from 7-EAA base pair with uridine and are well-accommodated within an A-form helix. We show that copper-catalyzed azide/alkyne cycloaddition (CuAAC) reactions with 7-EAA are sensitive to the RNA secondary structure context, with single-stranded sites reacting faster than duplex sites. 7-EAA and its triazole products are recognized in RNA template strands as adenosine by avian myoblastosis virus reverse transcriptase. In addition, 7-EAA in RNA is a substrate for an active site mutant of the RNA editing adenosine deaminase, ADAR2. These studies extend our understanding of the impact of these novel nucleobase analogues and set the stage for their use in probing RNA structure and metabolism.

The innate immune system relies on molecular sensors to detect distinctive molecular patterns, including viral double-stranded RNA (dsRNA), which triggers responses resulting in apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I), serving as a mechanism to distinguish self from non-self RNA and prevent aberrant immune activation. Loss-of-function mutations in the ADAR1 gene are one cause of Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. Although seven out of the eight AGS-associated mutations in ADAR1 occur within the catalytic domain of the ADAR1 protein, their specific effects on the catalysis of adenosine deamination remain poorly understood. In this study, we carried out a biochemical investigation of four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. These studies included adenosine deamination rate measurements with two different RNA substrates derived from human transcripts known to be edited by ADAR1 p110 (glioma-associated oncogene homologue 1 (hGli1), 5-hydroxytryptamine receptor 2C (5-HT2cR)). Our results indicate that AGS-associated mutations at two amino acid positions directly involved in stabilizing the base-flipped conformation of the ADAR-RNA complex (G1007R and R892H) had the most detrimental impact on catalysis. The K999N mutation, positioned near the RNA binding interface, altered catalysis contextually. Finally, the Y1112F mutation had small effects in each of the assays described here. These findings shed light on the differential effects of disease-associated mutations on adenosine deamination by ADAR1, thereby advancing our structural and functional understanding of ADAR1-mediated RNA editing.

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5' direction (5'-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5'-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5'-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5'-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5'-GA sites.

ADARs (adenosine deaminases acting on RNA) can be directed to sites in the transcriptome by complementary guide strands allowing for the correction of disease-causing mutations at the RNA level. However, ADARs show bias against editing adenosines with a guanosine 5' nearest neighbor (5'-GA sites), limiting the scope of this approach. Earlier studies suggested this effect arises from a clash in the RNA minor groove involving the 2-amino group of the guanosine adjacent to an editing site. Here we show that nucleosides capable of pairing with guanosine in a syn conformation enhance editing for 5'-GA sites. We describe the crystal structure of a fragment of human ADAR2 bound to RNA bearing a G:G pair adjacent to an editing site. The two guanosines form a Gsyn:Ganti pair solving the steric problem by flipping the 2-amino group of the guanosine adjacent to the editing site into the major groove. Also, duplexes with 2'-deoxyadenosine and 3-deaza-2'-deoxyadenosine displayed increased editing efficiency, suggesting the formation of a Gsyn:AH+anti pair. This was supported by X-ray crystallography of an ADAR complex with RNA bearing a G:3-deaza dA pair. This study shows how non-Watson-Crick pairing in duplex RNA can facilitate ADAR editing enabling the design of next generation guide strands for therapeutic RNA editing.

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

All messenger RNAs in eukaryotes are modified co-transcriptionally and post-transcriptionally. They are all capped at the 5'-end and polyadenylated at the 3'-end. However, many mRNAs are also found to be chemically modified internally for regulation of mRNA processing, translation, stability, and to recode the message. This review will briefly summarize the structural basis for formation of the two most common modifications found at internal sites in mRNAs; methylation and deamination. The structures of the enzymes that catalyze these modifications show structural similarity to other family members within each modifying enzyme class. RNA methyltransferases, including METTL3/METTL14 responsible for N6-methyladensosine (m6A) formation, share a common structural core and utilize S-adenosyl methionine as a methyl donor. RNA deaminases, including adenosine deaminases acting on RNA (ADARs), also share a common structural core and similar signature sequence motif with conserved residues used for binding zinc and catalyzing the deamination reaction. In spite of recent reports of high resolution structures for members of these two RNA-modifying enzyme families, a great deal remains to be uncovered for a complete understanding of the structural basis for mRNA modification. Of particular interest is the definition of factors that control modification site specificity.

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

Adenosine deaminases acting on RNA (ADARs) hydrolytically deaminate adenosines (A) in a wide variety of duplex RNAs and misregulation of editing is correlated with human disease. However, our understanding of reaction selectivity is limited. ADARs are modular enzymes with multiple double-stranded RNA binding domains (dsRBDs) and a catalytic domain. While dsRBD binding is understood, little is known about ADAR catalytic domain/RNA interactions. Here we use a recently discovered RNA substrate that is rapidly deaminated by the isolated human ADAR2 deaminase domain (hADAR2-D) to probe these interactions. We introduced the nucleoside analog 8-azanebularine (8-azaN) into this RNA (and derived constructs) to mechanistically trap the protein-RNA complex without catalytic turnover for EMSA and ribonuclease footprinting analyses. EMSA showed that hADAR2-D requires duplex RNA and is sensitive to 2'-deoxy substitution at nucleotides opposite the editing site, the local sequence and 8-azaN nucleotide positioning on the duplex. Ribonuclease V1 footprinting shows that hADAR2-D protects ∼ 23 nt on the edited strand around the editing site in an asymmetric fashion (∼ 18 nt on the 5' side and ∼ 5 nt on the 3' side). These studies provide a deeper understanding of the ADAR catalytic domain-RNA interaction and new tools for biophysical analysis of ADAR-RNA complexes.