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
We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.
- MeSH
- buněčné jádro metabolismus virologie MeSH
- DEAD-box RNA-helikasy metabolismus genetika MeSH
- genom virový MeSH
- HEK293 buňky MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus * genetika metabolismus fyziologie MeSH
- replikace viru genetika fyziologie MeSH
- RNA virová * metabolismus genetika MeSH
- RNA-helikasy metabolismus genetika MeSH
- sestavení viru * genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata 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
mRNAs containing premature stop codons are responsible for various genetic diseases as well as cancers. The truncated proteins synthesized from these aberrant mRNAs are seldom detected due to the nonsense-mediated mRNA decay (NMD) pathway. Such a surveillance mechanism detects most of these aberrant mRNAs and rapidly destroys them from the pool of mRNAs. Here, we implemented chemical cross-linking mass spectrometry (CLMS) techniques to trace novel biology consisting of protein-protein interactions (PPIs) within the NMD machinery. A set of novel complex networks between UPF2 (Regulator of nonsense transcripts 2), SMG1 (Serine/threonine-protein kinase SMG1), and SMG7 from the NMD pathway were identified, among which UPF2 was found as a connection bridge between SMG1 and SMG7. The UPF2 N-terminal formed most interactions with SMG7, and a set of residues emerged from the MIF4G-I, II, and III domains docked with SMG1 or SMG7. SMG1 mediated interactions with initial residues of UPF2, whereas SMG7 formed very few interactions in this region. Modelled structures highlighted that PPIs for UPF2 and SMG1 emerged from the well-defined secondary structures, whereas SMG7 appeared from the connecting loops. Comparing the influence of cancer-derived mutations over different CLMS sites revealed that variants in the PPIs for UPF2 or SMG1 have significant structural stability effects. Our data highlights the protein-protein interface of the SMG1, UPF2, and SMG7 genes that can be used for potential therapeutic approaches. Blocking the NMD pathway could enhance the production of neoantigens or internal cancer vaccines, which could provide a platform to design potential peptide-based vaccines.
Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.
- MeSH
- DNA-helikasy * genetika metabolismus MeSH
- genetická transkripce MeSH
- lidé MeSH
- multifunkční enzymy genetika metabolismus MeSH
- neurodegenerativní nemoci MeSH
- R-smyčka MeSH
- regulace genové exprese MeSH
- RNA-helikasy * metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- terminace genetické transkripce * MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.
- MeSH
- adenosintrifosfát metabolismus MeSH
- adenosintrifosfatasy * metabolismus MeSH
- dvouvláknová RNA metabolismus MeSH
- fosfáty metabolismus MeSH
- jednovláknová DNA * metabolismus MeSH
- lidé MeSH
- RNA-helikasy * metabolismus MeSH
- virové nestrukturální proteiny * metabolismus MeSH
- viry klíšťové encefalitidy * enzymologie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the 'regulatory loop (involved in mRNA binding)'. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: 'open (isoform_2; without insertion)' and 'closed (isoform_1; with insertion)'. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the 'open conformation'. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA-UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein-mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.
- MeSH
- alternativní sestřih * MeSH
- fosforylace MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- nonsense mediated mRNA decay * MeSH
- protein - isoformy MeSH
- regulace genové exprese * MeSH
- RNA-helikasy genetika metabolismus MeSH
- trans-aktivátory genetika metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.
- MeSH
- antivirové látky farmakologie MeSH
- COVID-19 MeSH
- farmakoterapie COVID-19 MeSH
- fluorescenční barviva chemie MeSH
- inhibitory proteas farmakologie MeSH
- koronavirové proteasy podobné papainu antagonisté a inhibitory chemie genetika metabolismus MeSH
- lidé MeSH
- preklinické hodnocení léčiv MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- RNA-helikasy antagonisté a inhibitory chemie genetika metabolismus MeSH
- RNA-viry enzymologie MeSH
- rychlé screeningové testy metody MeSH
- SARS-CoV-2 enzymologie MeSH
- serinové endopeptidasy chemie genetika metabolismus MeSH
- virové nestrukturální proteiny antagonisté a inhibitory chemie genetika metabolismus MeSH
- viry klíšťové encefalitidy enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Multisubunit cullin-RING ubiquitin ligase 4 (CRL4)-DCAF12 recognizes the C-terminal degron containing acidic amino acid residues. However, its physiological roles and substrates are largely unknown. Purification of CRL4-DCAF12 complexes revealed a wide range of potential substrates, including MOV10, an "ancient" RNA-induced silencing complex (RISC) complex RNA helicase. We show that DCAF12 controls the MOV10 protein level via its C-terminal motif in a proteasome- and CRL-dependent manner. Next, we generated Dcaf12 knockout mice and demonstrated that the DCAF12-mediated degradation of MOV10 is conserved in mice and humans. Detailed analysis of Dcaf12-deficient mice revealed that their testes produce fewer mature sperms, phenotype accompanied by elevated MOV10 and imbalance in meiotic markers SCP3 and γ-H2AX. Additionally, the percentages of splenic CD4+ T and natural killer T (NKT) cell populations were significantly altered. In vitro, activated Dcaf12-deficient T cells displayed inappropriately stabilized MOV10 and increased levels of activated caspases. In summary, we identified MOV10 as a novel substrate of CRL4-DCAF12 and demonstrated the biological relevance of the DCAF12-MOV10 pathway in spermatogenesis and T cell activation.
- MeSH
- aktivace lymfocytů fyziologie MeSH
- antigeny nádorové metabolismus MeSH
- buněčné linie MeSH
- CD4-pozitivní T-lymfocyty metabolismus MeSH
- HCT116 buňky MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- nádorové buněčné linie MeSH
- NKT buňky metabolismus MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- RNA-helikasy metabolismus MeSH
- spermatogeneze fyziologie MeSH
- ubikvitin metabolismus MeSH
- ubikvitinligasy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.
- MeSH
- adenosindeaminasa chemie genetika MeSH
- adenosinmonofosfát metabolismus MeSH
- bodová mutace genetika MeSH
- degenerace nervu patologie MeSH
- Drosophila melanogaster genetika imunologie MeSH
- editace RNA genetika MeSH
- katalýza MeSH
- lokomoce MeSH
- messenger RNA genetika metabolismus MeSH
- mozek metabolismus MeSH
- přirozená imunita genetika MeSH
- proteinové domény MeSH
- proteiny Drosophily chemie genetika metabolismus MeSH
- regulace genové exprese MeSH
- ribonukleasa III metabolismus MeSH
- RNA-helikasy metabolismus MeSH
- stárnutí patologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
