Viroids, small circular non-coding RNAs, act as infectious pathogens in higher plants, demonstrating high stability despite consisting solely of naked RNA. Their dependence of replication on host machinery poses the question of whether RNA modifications play a role in viroid biology. Here, we explore RNA modifications in the avocado sunblotch viroid (ASBVd) and the citrus exocortis viroid (CEVd), representative members of viroids replicating in chloroplasts and the nucleus, respectively, using LC - MS and Oxford Nanopore Technology (ONT) direct RNA sequencing. Although no modification was detected in ASBVd, CEVd contained approximately one m6A per RNA molecule. ONT sequencing predicted three m6A positions. Employing orthogonal SELECT method, we confirmed m6A in two positions A353 and A360, which are highly conserved among CEVd variants. These positions are located in the left terminal region of the CEVd rod-like structure where likely RNA Pol II and and TFIIIA-7ZF bind, thus suggesting potential biological role of methylation in viroid replication.

• Klíčová slova
• 6-methyladenosine, LC-MS, m6A SELECT, RNA modification, Viroid, direct RNA-seq,
• MeSH
• konformace nukleové kyseliny MeSH
• kruhová RNA * genetika   metabolismus   MeSH
• nemoci rostlin virologie   MeSH
• posttranskripční úpravy RNA * MeSH
• replikace viru MeSH
• RNA virová * genetika   metabolismus   chemie   MeSH
• sekvenční analýza RNA MeSH
• viroidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kruhová RNA * MeSH
• RNA virová * MeSH

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• adenosin analogy a deriváty   metabolismus   MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 genetika   metabolismus   MeSH
• anotace sekvence MeSH
• gen pro FTO genetika   metabolismus   MeSH
• genetická transkripce MeSH
• genová ontologie MeSH
• HEK293 buňky MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• messenger RNA genetika   metabolismus   MeSH
• methyltransferasy genetika   metabolismus   MeSH
• N-demethylasy genetika   metabolismus   MeSH
• nekódující RNA genetika   metabolismus   MeSH
• oprava DNA MeSH
• protein - isoformy genetika   metabolismus   MeSH
• replikace DNA MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• adenosin MeSH
• alfa-ketoglutarát-dependentní dioxygenasa, AlkB homolog 5 MeSH
• ALKBH5 protein, human MeSH Prohlížeč
• FTO protein, human MeSH Prohlížeč
• gen pro FTO MeSH
• jaderné proteiny MeSH
• messenger RNA MeSH
• methyltransferasy MeSH
• METTL16 protein, human MeSH Prohlížeč
• N-demethylasy MeSH
• N-methyladenosine MeSH Prohlížeč
• N(6),N(6)-dimethyladenosine MeSH Prohlížeč
• nekódující RNA MeSH
• PCIF1 protein, human MeSH Prohlížeč
• protein - isoformy MeSH

RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

• Klíčová slova
• DNA demethylation, DNA repair, RNA methylation, base excision repair, epigenetics,
• MeSH
• chromatin MeSH
• demetylace DNA * MeSH
• DNA metabolismus   MeSH
• metylace DNA MeSH
• oprava DNA MeSH
• PARP inhibitory * farmakologie   MeSH
• poškození DNA MeSH
• RNA genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• DNA MeSH
• PARP inhibitory * MeSH
• RNA MeSH

Tick-borne encephalitis virus (TBEV) is a leading cause of human neuroinfections in Europe and Northeast Asia. There are no antiviral therapies for treating TBEV infection. A series of nucleoside analogues was tested for the ability to inhibit the replication of TBEV in porcine kidney cells and human neuroblastoma cells. The interactions of three nucleoside analogues with viral polymerase were simulated using advanced computational methods. The nucleoside analogues 7-deaza-2'-C-methyladenosine (7-deaza-2'-CMA), 2'-C-methyladenosine (2'-CMA), and 2'-C-methylcytidine (2'-CMC) inhibited TBEV replication. These compounds showed dose-dependent inhibition of TBEV-induced cytopathic effects, TBEV replication (50% effective concentrations [EC50]of 5.1 ± 0.4 μM for 7-deaza-2'-CMA, 7.1 ± 1.2 μM for 2'-CMA, and 14.2 ± 1.9 μM for 2'-CMC) and viral antigen production. Notably, 2'-CMC was relatively cytotoxic to porcine kidney cells (50% cytotoxic concentration [CC50] of ∼50 μM). The anti-TBEV effect of 2'-CMA in cell culture diminished gradually after day 3 posttreatment. 7-Deaza-2'-CMA showed no detectable cellular toxicity (CC50 > 50 μM), and the antiviral effect in culture was stable for >6 days posttreatment. Computational molecular analyses revealed that compared to the other two compounds, 7-deaza-2'-CMA formed a large cluster near the active site of the TBEV polymerase. High antiviral activity and low cytotoxicity suggest that 7-deaza-2'-CMA is a promising candidate for further investigation as a potential therapeutic agent in treating TBEV infection.

• MeSH
• adenosin analogy a deriváty   chemie   farmakologie   MeSH
• antivirové látky chemie   farmakologie   MeSH
• buněčné linie MeSH
• cytidin analogy a deriváty   chemie   farmakologie   MeSH
• lidé MeSH
• nukleosidy chemie   farmakologie   MeSH
• prasata MeSH
• replikace viru účinky léků   MeSH
• tubercidin analogy a deriváty   chemie   farmakologie   MeSH
• viry klíšťové encefalitidy účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2'-C-methyladenosine MeSH Prohlížeč
• 2'-C-methylcytidine MeSH Prohlížeč
• 7-deaza-2'-C-methyladenosine MeSH Prohlížeč
• adenosin MeSH
• antivirové látky MeSH
• cytidin MeSH
• nukleosidy MeSH
• tubercidin MeSH

The supraoptic nuclei (SON), the hypothalamic release site of vasopressin and oxytocin, receive a non-glutamatergic, excitatory input from the caudal medulla that uses noradrenaline and ATP as neurotransmitters. Here, we studied the actions of extracellular ATP on SON neurons in hypothalamic slices isolated from the brains of 16- to 24-day-old rats. Whole-cell current clamp recordings performed 1-6 h after isolation showed that exogenous ATP application increased the frequency of action potentials and induced the depolarization of resting membranes. Voltage clamp recordings showed that ATP increased the frequency of GABAergic or glutamatergic spontaneous synaptic currents without changing their amplitude and evoked inward current (126±13 pA) in about 80% of SON neurons. The application of ATPγS and 2MeSATP mimicked the effects of ATP, but 2MeSADP, 2MeSAMP and αβmeATP had no effect. The P2X7 receptor agonist, BzATP, did not induce an inward current, but it increased intracellular calcium concentration in non-neuronal SON cells in slices. Suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) inhibited ATP-induced currents, whereas pH 6.5 and ivermectin, a specific allosteric modulator of the P2X4 receptor, potentiated ATP-induced currents. The P2Y1-selective antagonist, 2'-deoxy-N⁶-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), had no effect on ATP-induced responses. Quantitative real-time PCR showed that P2X2>P2X7>P2X4 purinergic receptor mRNAs were expressed in the SON tissue, but the levels of P2X1, P2X3, P2X5, P2X6, P2Y1, P2Y2 and P2Y12 mRNA were minor. These results show that SON neurons express functional presynaptic and extrasynaptic P2X2 and P2X4 receptors that modulate glutamate and GABA release and control the electrical excitability of SON neurons.

• MeSH
• adenosintrifosfát farmakologie   MeSH
• akční potenciály účinky léků   fyziologie   MeSH
• GABA metabolismus   MeSH
• krysa rodu Rattus MeSH
• kyselina glutamová metabolismus   MeSH
• metoda terčíkového zámku MeSH
• neurony účinky léků   metabolismus   MeSH
• nucleus supraopticus účinky léků   metabolismus   MeSH
• orgánové kultury - kultivační techniky MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potkani Wistar MeSH
• purinergní receptory P2X2 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• GABA MeSH
• kyselina glutamová MeSH
• purinergní receptory P2X2 MeSH