Adenosine deaminases acting on RNA (ADAR1 and ADAR2) are human RNA-editing adenosine deaminases responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. Since inosine is recognized during translation as guanosine, this often results in the expression of protein sequences different from those encoded in the genome. While our knowledge of the ADAR2 structure and catalytic mechanism has grown over the years, our knowledge of ADAR1 has lagged. This is due, at least in part, to the lack of well defined, small RNA substrates useful for mechanistic studies of ADAR1. Here, we describe an ADAR1 substrate RNA that can be prepared by a combination of chemical synthesis and enzymatic ligation. Incorporation of adenosine analogs into this RNA and analysis of the rate of ADAR1 catalyzed deamination revealed similarities and differences in the way the ADARs recognize the edited nucleotide. Importantly, ADAR1 is more dependent than ADAR2 on the presence of N7 in the edited base. This difference between ADAR1 and ADAR2 appears to be dependent on the identity of a single amino acid residue near the active site. Thus, this work provides an important starting point in defining mechanistic differences between two functionally distinct human RNA editing ADARs.

Adenosine deaminases that act on RNA (ADARs) can be directed to predetermined sites in transcriptomes by forming duplex structures with exogenously delivered guide RNAs (gRNAs). They can then catalyze the hydrolytic deamination of adenosine to inosine in double stranded RNA, which is read as guanosine during translation. High resolution structures of ADAR2-RNA complexes revealed a unique conformation for the nucleotide in the guide strand base paired to the editing site's 5' nearest neighbor (-1 position). Here we describe the effect of 16 different nucleoside analogs at this position in a gRNA that targets a 5'-UA̲-3' site. We found that several analogs increase editing efficiency for both catalytically active human ADARs. In particular, 2'-deoxynebularine (dN) increased the ADAR1 and ADAR2 in vitro deamination rates when at the -1 position of gRNAs targeting the human MECP2 W104X site, the mouse IDUA W392X site, and a site in the 3'-UTR of human ACTB. Furthermore, a locked nucleic acid (LNA) modification at the -1 position was found to eliminate editing. When placed -1 to a bystander editing site in the MECP2 W104X sequence, bystander editing was eliminated while maintaining on-target editing. In vitro trends for four -1 nucleoside analogs were validated by directed editing of the MECP2 W104X site expressed on a reporter transcript in human cells. This work demonstrates the importance of the -1 position of the gRNA to ADAR editing and discloses nucleoside analogs for this site that modulate ADAR editing efficiency.

A number of genes have been identified that are highly expressed in the post-infective L3 stage of the filarial parasite, Brugia pahangi. Amongst these was a cDNA with homology to the cytidine deaminase (CDD) gene family. Phylogenetic analysis of the various cytosine nucleoside deaminases suggest that Brugia pahangi CDD evolved with significant divergence from the RNA editing family. In order to characterize its function, we have expressed Brugia pahangi CDD in bacteria as a chimera with maltose-binding protein (MBP). Biochemical analysis demonstrates the MBP-CDD fusion protein functions as an authentic cytidine deaminase with an obligate requirement for zinc. In addition to cytidine deaminase activity, however, the fusion protein demonstrates RNA binding activity with specificity for AU-rich sequences and was found to bind an RNA template spanning the edited site of mammalian apolipoprotein B (apoB) mRNA. This RNA binding activity was not found in two different recombinant bacterial CDD proteins. In vitro RNA editing assays revealed that MBP-CDD failed to mediate cytidine deamination of a mammalian apoB RNA template. Furthermore, binding of MBP-CDD to the apoB RNA did not inhibit in vitro editing of this template by apobec-1. The data suggest that the cytosine nucleoside deaminases and RNA editing deaminases have acquired different mechanisms of binding to an AU-rich RNA template, presumably with different functional implications.

Codon usage bias is a universal feature of all genomes and plays an important role in regulating protein expression levels. Modification of adenosine to inosine at the tRNA anticodon wobble position (I34) by adenosine deaminases (ADATs) is observed in all eukaryotes and has been proposed to explain the correlation between codon usage and tRNA pool. However, how the tRNA pool is affected by I34 modification to influence codon usage-dependent gene expression is unclear. Using Neurospora crassa as a model system, by combining molecular, biochemical and bioinformatics analyses, we show that silencing of adat2 expression severely impaired the I34 modification levels for the ADAT-related tRNAs, resulting in major ADAT-related tRNA profile changes and reprogramming of translation elongation kinetics on ADAT-related codons. adat2 silencing also caused genome-wide codon usage-biased ribosome pausing on mRNAs and proteome landscape changes, leading to selective translational repression or induction of different mRNAs. The induced expression of CPC-1, the Neurospora ortholog of yeast GCN4p, mediates the transcriptional response after adat2 silencing and amino acid starvation. Together, our results demonstrate that the tRNA I34 modification by ADAT plays a major role in driving codon usage-biased translation to shape proteome landscape.

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-β (IFN-β) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-β. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-β. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-β production.

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.

Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/β receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 μM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα.

The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (-)HIV-1 DNA. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an ¹⁹⁰NPM¹⁹² motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F ¹⁹¹Pro to ¹⁹¹Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F ¹⁹⁰NGM¹⁹² could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif (APOBEC3F, 5'TTC; APOBEC3G 5'CCC) influences the mutagenic and gene inactivation potential of an APOBEC3 enzyme.

Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of Trypanosoma brucei CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2'-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs T. brucei proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for de novo thymidylate biosynthesis. This observation contrasts with the existence in T. brucei of a dimeric deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, T. brucei dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in T. brucei, CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism.IMPORTANCE Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in Trypanosoma brucei exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant T. brucei CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5'-triphosphate nucleotidohydrolase does not have a prominent role in de novo dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway.

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.