Adar2-/- mice are a widely used model for studying the physiological consequences of reduced RNA editing. These mice are viable only when the Q/R editing site of the Gria2 subunit of the AMPA receptor is constitutively mutated to the codon for arginine, and Gria2R/R mice often serve as the sole control for Adar2-/- mice. Our study aimed to investigate whether ADAR2 inactivity and the Gria2R/R phenotype affect the rhythmicity of the circadian clock gene pattern and the expression of Gria1 and Gria2 subunits in the suprachiasmatic nucleus (SCN), hippocampus, parietal cortex and liver. Our data show that Gria2R/R mice completely lost circadian rhythmicity in the hippocampus compared to Adar2-/- mice. Compared to C57BL/6J mice, the expression profiles in the hippocampus and parietal cortex of Gria2R/R mice differ to the same extent as in Adar2-/-. No alterations were detected in the circadian profiles in the livers. These data suggest that the natural gradual postnatal increase in the editing of the Q/R site of the Gria2 subunit may be important for the development of circadian clockwork in some brain structures, and the use of Gria2R/R mice as the only control to Adar2-/- mice in the experiments dependent on the hippocampus and parietal cortex should therefore be considered.
- MeSH
- adenosindeaminasa genetika metabolismus MeSH
- cirkadiánní rytmus * MeSH
- exprese genu MeSH
- hipokampus metabolismus MeSH
- mozek * metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nucleus suprachiasmaticus metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.
Somatic hypermutation (SHM) drives the genetic diversity of Ig genes in activated B cells and supports the generation of Abs with increased affinity for Ag. SHM is targeted to Ig genes by their enhancers (diversification activators [DIVACs]), but how the enhancers mediate this activity is unknown. We show using chicken DT40 B cells that highly active DIVACs increase the phosphorylation of RNA polymerase II (Pol II) and Pol II occupancy in the mutating gene with little or no accompanying increase in elongation-competent Pol II or production of full-length transcripts, indicating accumulation of stalled Pol II. DIVAC has similar effect also in human Ramos Burkitt lymphoma cells. The DIVAC-induced stalling is weakly associated with an increase in the detection of ssDNA bubbles in the mutating target gene. We did not find evidence for antisense transcription, or that DIVAC functions by altering levels of H3K27ac or the histone variant H3.3 in the mutating gene. These findings argue for a connection between Pol II stalling and cis-acting targeting elements in the context of SHM and thus define a mechanistic basis for locus-specific targeting of SHM in the genome. Our results suggest that DIVAC elements render the target gene a suitable platform for AID-mediated mutation without a requirement for increasing transcriptional output.
- MeSH
- aktivace lymfocytů MeSH
- Burkittův lymfom genetika imunologie MeSH
- cytidindeaminasa genetika MeSH
- genetická transkripce MeSH
- imunoglobuliny genetika metabolismus MeSH
- kur domácí MeSH
- lidé MeSH
- mutace genetika MeSH
- mutageneze cílená MeSH
- podskupiny B-lymfocytů imunologie MeSH
- ptačí proteiny genetika metabolismus MeSH
- RNA-polymerasa II genetika metabolismus MeSH
- rozmanitost protilátek MeSH
- somatická hypermutace imunoglobulinových genů MeSH
- zesilovače transkripce genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Squamous cell carcinoma of the penis (PSC) is a rare disease with limited information on the molecular events leading to malignant transformation. In a third of PSC cases, presence of human papilloma virus (HPV) is found. The APOBEC3 family of proteins is known to play a significant role in defense against HPV infection, but their role in PSC is largely unknown. In this study, we aim to assess mRNA expression levels of APOBEC3 family members in HPV+ and HPV- PSC to get insight into their association with clinicopathological features and to evaluate their prognostic impact. Expression levels of six APOBEC3 family members in tissue from 50 patients with PSC were determined by RT-PCR and correlated with clinical and histopathological features. Lower expression of APOBEC3A, APOBEC3B, and APOBEC3C was observed in advanced PSC stages. Except for APOBEC3D, HPV+ samples showed higher expression of APOBEC3s compared to HPV- samples. In univariate analyses, APOBEC3A and APOBEC3C expression tended to be associated with disease-free survival and APOBEC3A expression with overall survival; however, multivariable analyses failed to confirm these associations with outcome. More extensive external validation and functional laboratory studies are needed to evaluate further their role in PSC development and progression.
- MeSH
- cytidindeaminasa genetika MeSH
- deaminasy APOBEC MeSH
- infekce papilomavirem * komplikace genetika MeSH
- lidé MeSH
- Papillomaviridae genetika MeSH
- penis patologie MeSH
- prognóza MeSH
- spinocelulární karcinom * patologie MeSH
- vedlejší histokompatibilní antigeny genetika MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Multiple sclerosis (MS) is a highly disabling, progressive neurodegenerative disease with no curative treatment available. Although significant progress has been made in understanding how MS develops, there remain aspects of disease pathogenesis that are yet to be fully elucidated. In this regard, studies have shown that dysfunctional adenosinergic signaling plays a pivotal role, as patients with MS have altered levels adenosine (ADO), adenosine receptors and proteins involved in the generation and termination of ADO signaling, such as CD39 and adenosine deaminase (ADA). We have therefore performed a literature review regarding the involvement of the adenosinergic system in the development of MS and propose mechanisms by which the modulation of this system can support drug development and repurposing.
- MeSH
- adenosin imunologie MeSH
- adenosindeaminasa imunologie MeSH
- apyrasa imunologie MeSH
- lidé MeSH
- neurodegenerativní nemoci * etiologie imunologie terapie MeSH
- purinergní receptory P1 * imunologie MeSH
- roztroušená skleróza * etiologie imunologie terapie MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
ADAR1 edits adenosines to inosines in cellular double-stranded (ds)RNA, thereby preventing aberrant activation of antiviral dsRNA sensors, as well as interferon (IFN) induction in Aicardi-Goutières syndrome (AGS) encephalopathy. Recently, Nakahama et al., Tang et al., Maurano et al., and de Reuver et al. demonstrated that Adar1 Zα domain-mutant mice show aberrant MDA5 and PKR activation, developing encephalopathies; short Z-RNA patches within cellular dsRNA are unexpectedly crucial in causing aberrant antiviral responses.
- MeSH
- adenosindeaminasa * genetika metabolismus MeSH
- antivirové látky MeSH
- autoimunitní nemoci nervového systému * genetika MeSH
- dvouvláknová RNA MeSH
- editace RNA MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Adenosine-to-inosine RNA editing is a co-transcriptional/post-transcriptional modification of double-stranded RNA, catalysed by one of two active adenosine deaminases acting on RNA (ADARs), ADAR1 and ADAR2. ADARB1 encodes the enzyme ADAR2 that is highly expressed in the brain and essential to modulate the function of glutamate and serotonin receptors. Impaired ADAR2 editing causes early onset progressive epilepsy and premature death in mice. In humans, ADAR2 dysfunction has been very recently linked to a neurodevelopmental disorder with microcephaly and epilepsy in four unrelated subjects. METHODS: We studied three children from two consanguineous families with severe developmental and epileptic encephalopathy (DEE) through detailed physical and instrumental examinations. Exome sequencing (ES) was used to identify ADARB1 mutations as the underlying genetic cause and in vitro assays with transiently transfected cells were performed to ascertain the impact on ADAR2 enzymatic activity and splicing. RESULTS: All patients showed global developmental delay, intractable early infantile-onset seizures, microcephaly, severe-to-profound intellectual disability, axial hypotonia and progressive appendicular spasticity. ES revealed the novel missense c.1889G>A, p.(Arg630Gln) and deletion c.1245_1247+1 del, p.(Leu415PhefsTer14) variants in ADARB1 (NM_015833.4). The p.(Leu415PhefsTer14) variant leads to incorrect splicing resulting in frameshift with a premature stop codon and loss of enzyme function. In vitro RNA editing assays showed that the p.(Arg630Gln) variant resulted in a severe impairment of ADAR2 enzymatic activity. CONCLUSION: In conclusion, these data support the pathogenic role of biallelic ADARB1 variants as the cause of a distinctive form of DEE, reinforcing the importance of RNA editing in brain function and development.
- MeSH
- adenosindeaminasa genetika metabolismus MeSH
- alely MeSH
- dítě MeSH
- dvouvláknová RNA metabolismus MeSH
- editace RNA MeSH
- epilepsie enzymologie genetika MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mutace MeSH
- nemoci mozku enzymologie genetika metabolismus MeSH
- neurovývojové poruchy enzymologie genetika MeSH
- pokrevní příbuzenství MeSH
- předškolní dítě MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- rodokmen MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
The genomic diversity of SARS-CoV-2 has been a focus during the ongoing COVID-19 pandemic. Here, we analyzed the distribution and character of emerging mutations in a data set comprising more than 95,000 virus genomes covering eight major SARS-CoV-2 lineages in the GISAID database, including genotypes arising during COVID-19 therapy. Globally, the C>U transitions and G>U transversions were the most represented mutations, accounting for the majority of single-nucleotide variations. Mutational spectra were not influenced by the time the virus had been circulating in its host or medical treatment. At the amino acid level, we observed about a 2-fold excess of substitutions in favor of hydrophobic amino acids over the reverse. However, most mutations constituting variants of interests of the S-protein (spike) lead to hydrophilic amino acids, counteracting the global trend. The C>U and G>U substitutions altered codons towards increased amino acid hydrophobicity values in more than 80% of cases. The bias is explained by the existing differences in the codon composition for amino acids bearing contrasting biochemical properties. Mutation asymmetries apparently influence the biochemical features of SARS CoV-2 proteins, which may impact protein-protein interactions, fusion of viral and cellular membranes, and virion assembly.
- MeSH
- alely MeSH
- aminokyseliny chemie genetika MeSH
- COVID-19 virologie MeSH
- deaminasy APOBEC MeSH
- fylogeneze MeSH
- genetická variace MeSH
- genom virový * MeSH
- genotyp MeSH
- glykoprotein S, koronavirus chemie genetika MeSH
- hydrofobní a hydrofilní interakce * MeSH
- interakce hostitele a patogenu MeSH
- interakční proteinové domény a motivy MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- molekulární evoluce MeSH
- mutace * MeSH
- SARS-CoV-2 genetika MeSH
- substituce aminokyselin MeSH
- vazba proteinů MeSH
- virové proteiny chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.
Modified bases act as marks on cellular RNAs so that they can be distinguished from foreign RNAs, reducing innate immune responses to endogenous RNA. In humans, mutations giving reduced levels of one base modification, adenosine-to-inosine deamination, cause a viral infection mimic syndrome, a congenital encephalitis with aberrant interferon induction. These Aicardi-Goutières syndrome 6 mutations affect adenosine deaminase acting on RNA 1 (ADAR1), which generates inosines in endogenous double-stranded (ds)RNA. The inosine base alters dsRNA structure to prevent aberrant activation of antiviral cytosolic helicase RIG-I-like receptors. We review how effects of inosines, ADARs, and other modified bases have been shown to be important in innate immunity and cancer.
