The RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) is essential for correct functioning of innate immune responses. The ADAR1p110 isoform is mainly nuclear and ADAR1p150, which is interferon (IFN) inducible, is predominately cytoplasmic. Using three different methods - co-immunoprecipitation (co-IP) of endogenous ADAR1, Strep-tag co-IP and BioID with individual ADAR1 isoforms - a comprehensive interactome was generated during both homeostasis and the IFN response. Both known and novel interactors as well as editing regulators were identified. Nuclear proteins were detected as stable interactors with both ADAR1 isoforms. In contrast, BioID identified distinct protein networks for each ADAR1 isoform, with nuclear components observed with ADAR1p110 and components of cytoplasmic cellular condensates with ADAR1p150. RNase A digestion distinguished between distal and proximal interactors, as did a double-stranded RNA (dsRNA)-binding mutant of ADAR1 which demonstrated the importance of dsRNA binding for ADAR1 interactions. IFN treatment did not affect the core ADAR1 interactomes but resulted in novel interactions, the majority of which are proximal interactions retained after RNase A treatment. Short treatment with high molecular weight poly(I:C) during the IFN response resulted in dsRNA-binding-dependent changes in the proximal protein network of ADAR1p110 and association of the ADAR1p150 proximal protein network with some components of antiviral stress granules.

• MeSH
• adenosindeaminasa * metabolismus   genetika   MeSH
• buněčné jádro * metabolismus   MeSH
• cytoplazma * metabolismus   MeSH
• dvouvláknová RNA metabolismus   genetika   MeSH
• editace RNA MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• interferony metabolismus   genetika   MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• poly I-C farmakologie   MeSH
• protein - isoformy * metabolismus   genetika   MeSH
• proteiny vázající RNA * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

G-quadruplexes (G4s) formed within RNA are emerging as promising targets for therapeutic intervention in cancer, neurodegenerative disorders and infectious diseases. Sequences containing a succession of short GG blocks, or uneven G-tract lengths unable to form three-tetrad G4s (GG motifs), are overwhelmingly more frequent than canonical motifs involving multiple GGG blocks. We recently showed that DNA is not able to form stable two-tetrad intramolecular parallel G4s. Whether RNA GG motifs can form intramolecular G4s under physiological conditions and play regulatory roles remains a burning question. In this study, we performed a systematic analysis and experimental evaluation of a number of biologically important RNA regions involving RNA GG motifs. We show that most of these motifs do not form stable intramolecular G4s but need to dimerize to form stable G4 structures. The strong tendency of RNA GG motif G4s to associate may participate in RNA-based aggregation under conditions of cellular stress.

• MeSH
• dimerizace MeSH
• G-kvadruplexy * MeSH
• genetická transkripce MeSH
• lidé MeSH
• nukleotidové motivy * MeSH
• RNA * chemie   metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-helikasy metabolismus   genetika   MeSH
• DNA * metabolismus   chemie   MeSH
• dvouřetězcové zlomy DNA * MeSH
• fosforylace MeSH
• homeostáza genetika   MeSH
• lidé MeSH
• oprava DNA * MeSH
• proteinfosfatasa 2 metabolismus   genetika   MeSH
• R-smyčka MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• RNA-polymerasa II * metabolismus   MeSH
• RNA * metabolismus   genetika   chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The ribosome, owing to its exceptional conservation, harbours a remarkable molecular fossil known as the protoribosome. It surrounds the peptidyl transferase center (PTC), responsible for peptide bond formation. While previous studies have demonstrated the PTC activity in RNA alone, our investigation reveals the intricate roles of the ribosomal protein fragments (rPeptides) within the ribosomal core. This research highlights the significance of rPeptides in stability and coacervation of two distinct protoribosomal evolutionary stages. The 617nt 'big' protoribosome model, which associates with rPeptides specifically, exhibits a structurally defined and rigid nature, further stabilized by the peptides. In contrast, the 136nt 'small' model, previously linked to peptidyltransferase activity, displays greater structural flexibility. While this construct interacts with rPeptides with lower specificity, they induce coacervation of the 'small' protoribosome across a wide concentration range, which is concomitantly dependent on the RNA sequence and structure. Moreover, these conditions protect RNA from degradation. This phenomenon suggests a significant evolutionary advantage in the RNA-protein interaction at the early stages of ribosome evolution. The distinct properties of the two protoribosomal stages suggest that rPeptides initially provided compartmentalization and prevented RNA degradation, preceding the emergence of specific RNA-protein interactions crucial for the ribosomal structural integrity.

• MeSH
• konformace nukleové kyseliny MeSH
• molekulární modely MeSH
• peptidy chemie   metabolismus   MeSH
• peptidyltransferasy metabolismus   chemie   MeSH
• ribozomální proteiny * metabolismus   chemie   MeSH
• ribozomy * metabolismus   MeSH
• RNA metabolismus   chemie   MeSH
• stabilita RNA MeSH
• Publikační typ
• časopisecké články MeSH

Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair.

• MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• jednovláknová DNA * metabolismus   genetika   MeSH
• lidé MeSH
• multiproteinové komplexy MeSH
• mutace MeSH
• rekombinasa Rad51 * metabolismus   genetika   MeSH
• replikace DNA * genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

• MeSH
• "flap" endonukleasy metabolismus   genetika   MeSH
• DEAD-box RNA-helikasy * metabolismus   genetika   MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• DNA-helikasy * metabolismus   genetika   MeSH
• DNA-ligasa ATP metabolismus   genetika   MeSH
• DNA metabolismus   genetika   MeSH
• endonukleasy * metabolismus   genetika   MeSH
• genetická transkripce MeSH
• lidé MeSH
• multifunkční enzymy * metabolismus   genetika   MeSH
• R-smyčka * MeSH
• replikace DNA * MeSH
• RNA-helikasy * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.

• MeSH
• DNA * chemie   genetika   MeSH
• G-kvadruplexy * MeSH
• inzulin * chemie   genetika   MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• nukleotidové motivy MeSH
• polymorfismus genetický MeSH
• promotorové oblasti (genetika) * MeSH
• reportérové geny MeSH
• sekvence nukleotidů MeSH
• tandemové repetitivní sekvence genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

• MeSH
• adenosindeaminasa * metabolismus   genetika   MeSH
• aktivace enzymů MeSH
• buňky A549 MeSH
• dvouvláknová RNA * metabolismus   MeSH
• kinasa eIF-2 * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• proteinové domény MeSH
• proteiny vázající RNA * metabolismus   genetika   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

We currently lack antivirals for most human viruses. In a quest for new molecules, focusing on viral RNA, instead of viral proteins, can represent a promising strategy. In this study, new inhibitors were identified starting from a published crystal structure of the tertiary SARS-CoV-2 RNA involved in the -1 programmed ribosomal frameshift. The pseudoknot structure was refined, and a virtual screening was performed using the repository of binders to the nucleic acid library, taking into consideration RNA flexibility. Hit compounds were validated against the wild-type virus and with a dual-luciferase assay measuring the frameshift efficiency. Several active molecules were identified. Our study reveals new inhibitors of SARS-CoV-2 but also highlights the feasibility of targeting RNA starting from virtual screening, a strategy that could be broadly applied to drug development.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• molekulární modely MeSH
• preklinické hodnocení léčiv * metody   MeSH
• RNA virová * metabolismus   antagonisté a inhibitory   genetika   MeSH
• SARS-CoV-2 * účinky léků   MeSH
• simulace molekulového dockingu MeSH
• uživatelské rozhraní počítače MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Armadillo repeat-containing proteins (ARMCs) are a large family found throughout eukaryotes, which play prominent roles in cell adhesion, signaling and cytoskeletal regulation. The ARMC6 protein is highly conserved in primates, including humans, but to date does not have a clear function beyond initial hints of a link to cancer and telomerase activity. We report here in vitro experiments showing ARMC6 binding to DNA promoter sequences from several cancer-related genes (e.g., EGFR, VEGF and c-MYC), and also to the telomeric RNA repeat (TERRA). ARMC6 binding activity appears to recognize G-quadruplex motifs, which are being increasingly implicated as structure-based protein binding sites in chromosome maintenance and repair. In vivo investigation of ARMC6 function revealed that when this protein is overexpressed in human cell lines, there is different expression of genes connected with oncogenic pathways and those implicated in downstream non-canonical telomerase pathways (e.g., VEGF, hTERT, c-MYC, ESM1, MMP3). ARMC6 is already known to interact with human shelterin protein TRF2 and telomerase. The protein binds G-quadruplex structures and does so preferentially to RNA over DNA. As such, this protein may be an example of how a non-canonical nucleic acid structural motif allows mediation between gene regulation and telomeric chromatin rearrangement pathways.

• MeSH
• DNA vazebné proteiny MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory genetika   metabolismus   MeSH
• promotorové oblasti (genetika) * MeSH
• proteiny s doménou armadillo * metabolismus   genetika   MeSH
• regulace genové exprese u nádorů MeSH
• RNA metabolismus   genetika   MeSH
• telomerasa metabolismus   genetika   MeSH
• telomery * metabolismus   MeSH
• transkripční faktory MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH