DEAD-box RNA helicase, a putative subunit of the mitochondrial ribosome of Trypanosoma brucei, has been down-regulated in the procyclic and bloodstream stage by RNA interference. Although ablation of the transcript leads to a week growth phenotype in the procyclic cells, the protein does not seem to be essential for mitochondrial translation under standard cultivation conditions, as shown by an assay that allows visualization of the de novo synthesized proteins. Furthermore, we show that synthesis of cytochrome c oxidase subunit I and cytochrome b does not occur in the mitochondrion of the bloodstream stage.

• MeSH
• cytochromy b biosyntéza   MeSH
• DEAD-box RNA-helikasy genetika   fyziologie   MeSH
• mitochondrie fyziologie   MeSH
• proteosyntéza fyziologie   MeSH
• respirační komplex IV biosyntéza   MeSH
• ribozomy enzymologie   genetika   MeSH
• RNA interference MeSH
• Trypanosoma brucei brucei enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytochromy b MeSH
• DEAD-box RNA-helikasy MeSH
• respirační komplex IV 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.

• Klíčová slova
• ATPase, RNA helicase, crystal structure, enzyme kinetics, flavivirus, molecular dynamics, nonstructural protein 3, tick-borne encephalitis virus, viral protein,
• 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
• Názvy látek
• adenosintrifosfát MeSH
• adenosintrifosfatasy * MeSH
• dvouvláknová RNA MeSH
• fosfáty MeSH
• jednovláknová DNA * MeSH
• NS3 protein, flavivirus MeSH Prohlížeč
• RNA-helikasy * MeSH
• virové nestrukturální proteiny * MeSH

The DEAD box p68 RNA helicase (DDX5) is required to manipulate RNA structures implicated in mRNA/rRNA processing and transcript export, and acts as a co-activator for a range of transcription factors. Previous research has indicated that p68 RNA helicase may also be important in tumour development. Wild-type HeLa and stable HeLa (clone 13) cell cultures containing RNAi-mediated depletion of p68 RNA helicase induced by doxycycline (DOX) were used to study how the p68 RNA helicase affects the mTOR cell signalling pathway. Relevant results were repeated using transient transfection with pSuper/pSuper-p68 RNA helicase, containing RNAi-mediated depletion of p68 RNA helicase, to avoid DOX interference. Here we provide strong evidence for the participation of p68 RNA helicase in mTOR regulation. In detail, depletion of this helicase decreases cell growth and activates the mTOR/MDM2 cell survival mechanism, which ultimately leads to inhibition of the pro-apoptotic activity. p68 RNA helicase downregulation strongly stimulates 4E-BP1 phosphorylation, thereby provoking activation of cap-dependent translation. In contrast, the IRES-dependent translation of c-myc is reduced when p68 RNA helicase is depleted, thus indicating that at least this specific translation requires p68 RNA helicase activity to manipulate the complex 5' end of this mRNA. Interestingly, p68 RNA helicase depletion decreases cell growth while activating the mTOR/MDM2 cell survival mechanism. As MDM2 is a known negative regulator of p53, we infer that the activation of the cell survival mechanism may result in inhibition of the pro-apoptotic factor p53. Finally, p68 RNA helicase depletion activates capdependent translation and inhibits c-MYC IRES-mediated translation.

• MeSH
• buněčný cyklus genetika   fyziologie   MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• fosforylace genetika   fyziologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• proliferace buněk fyziologie   MeSH
• protoonkogenní proteiny c-mdm2 genetika   metabolismus   MeSH
• RNA interference MeSH
• TOR serin-threoninkinasy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• MDM2 protein, human MeSH Prohlížeč
• MTOR protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• TOR serin-threoninkinasy 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
• Názvy látek
• "flap" endonukleasy MeSH
• DEAD-box RNA-helikasy * MeSH
• DNA vazebné proteiny * MeSH
• DNA-helikasy * MeSH
• DNA-ligasa ATP MeSH
• DNA MeSH
• endonukleasy * MeSH
• multifunkční enzymy * MeSH
• MUS81 protein, human MeSH Prohlížeč
• RNA-helikasy * MeSH
• SETX protein, human MeSH Prohlížeč

KEY MESSAGE: In Physcomitrium patens, PpRH1/PpRH2 are GUCT-domain-containing DEAD-BOX RNA helicases localize to the nucleus. They are implicated in cell and tissue development in all stages of the moss life cycle. ABSTRACT: The DEAD-box-containing RNA helicase family encompasses a large and functionally important group of enzymes involved in cellular processes committed to the metabolism of RNA, including its transcription, processing, transport, translation and decay. Studies indicate this protein family has implied roles in plant vegetative and reproductive developmental processes as well as response to environmental stresses such has cold and high salinity. We focus here on a small conserved sub-group of GUCT domain-containing RNA helicase in the moss Physcomitrium patens. Phylogenetic analysis shows that RNA helicases containing the GUCT domain form a distinct conserved clade across the green lineage. In this clade, the P. patens genome possesses two closely related paralogues RNA helicases predicted to be nuclear, PpRH1 and PpRH2. Using in-locus gene fluorescent tagging we show that PpRH1 is localized to the nucleus in protonema. Analysis of PpRH1 and PpRH2 deletions, individually and together, indicates their potential roles in protonema, gametophore and sporophyte cellular and tissue development in P. patens. Additionally, the ultrastructural analysis of phyllid chloroplasts in Δrh2 and Δrh1/2 shows distinct starch granule accumulation under standard growth conditions associated with changes in photosynthetic activity parameters. We could not detect effects of either temperature or stress on protonema growth or PpRH1 and PpRH2 expression. Together, these results suggest that nuclear GUCT-containing RNA helicases play a role primarily in developmental processes directly or indirectly linked to photosynthesis activity in the moss P. patens. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11103-021-01152-w.

• Klíčová slova
• Development, Gametophyte, Physcomitrium patens, RNA helicase, Sporophyte, Starch accumulation,
• MeSH
• buněčné jádro metabolismus   MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• mechy genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• RNA-helikasy MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• škrob metabolismus   MeSH
• zárodečné buňky rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• RNA-helikasy MeSH
• rostlinné proteiny MeSH
• škrob MeSH

The mitochondrial RNA binding complex 1 (MRB1) is a recently discovered complex of proteins associated with the TbRGG1 and TbRGG2 proteins in Trypanosoma brucei. Based on the phenotype caused by down-regulation of these two proteins, it was proposed to play an unspecified role in RNA editing. RNAi silencing of three newly characterized protein subunits, guide RNA associated proteins (GAPs) 1 and 2 as well as a predicted DExD/H-box RNA helicase, show they are essential for cell growth in the procyclic stage. Furthermore, their down-regulation leads to inhibition of editing in only those mRNAs for which minicircle-encoded guide (g) RNAs are required. However, editing remains unaffected when the maxicircle-encoded cis-acting gRNA is employed. Interestingly, all three proteins are necessary for the expression of the minicircle-encoded gRNAs. Moreover, down-regulation of a fourth assayed putative MRB1 subunit, Nudix hydrolase, does not appear to destabilize gRNAs, and down-regulation of this protein has a general impact on the stability of maxicircle-encoded RNAs. GAP1 and 2 are also essential for the survival of the bloodstream stage, in which the gRNAs become eliminated upon depletion of either protein. Immunolocalization revealed that GAP1 and 2 are concentrated into discrete spots along the mitochondrion, usually localized in the proximity of the kinetoplast. Finally, we demonstrate that the same mtRNA polymerase known to transcribe the maxicircle mRNAs may also have a role in expression of the minicircle-encoded gRNAs.

• MeSH
• DEAD-box RNA-helikasy metabolismus   MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• guide RNA, Kinetoplastida genetika   MeSH
• mitochondriální proteiny metabolismus   MeSH
• NUDIX hydrolasy MeSH
• proteiny vázající RNA metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• pyrofosfatasy metabolismus   MeSH
• RNA mitochondriální MeSH
• RNA protozoální genetika   MeSH
• RNA genetika   MeSH
• Trypanosoma brucei brucei genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• DNA řízené RNA-polymerasy MeSH
• guide RNA, Kinetoplastida MeSH
• mitochondriální proteiny MeSH
• proteiny vázající RNA MeSH
• protozoální proteiny MeSH
• pyrofosfatasy MeSH
• RNA mitochondriální MeSH
• RNA protozoální MeSH
• RNA 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.

• Klíčová slova
• DEAH-box RNA helicase, DHX15, G-patch, gRNA packaging, retrovirus,
• 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
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• DHX15 protein, human MeSH Prohlížeč
• RNA virová * MeSH
• RNA-helikasy MeSH

Collisions between replication and transcription machineries represent a significant source of genomic instability. RECQ5 DNA helicase binds to RNA-polymerase (RNAP) II during transcription elongation and suppresses transcription-associated genomic instability. Here, we show that RECQ5 also associates with RNAPI and enforces the stability of ribosomal DNA arrays. We demonstrate that RECQ5 associates with transcription complexes in DNA replication foci and counteracts replication fork stalling in RNAPI- and RNAPII-transcribed genes, suggesting that RECQ5 exerts its genome-stabilizing effect by acting at sites of replication-transcription collisions. Moreover, RECQ5-deficient cells accumulate RAD18 foci and BRCA1-dependent RAD51 foci that are both formed at sites of interference between replication and transcription and likely represent unresolved replication intermediates. Finally, we provide evidence for a novel mechanism of resolution of replication-transcription collisions wherein the interaction between RECQ5 and proliferating cell nuclear antigen (PCNA) promotes RAD18-dependent PCNA ubiquitination and the helicase activity of RECQ5 promotes the processing of replication intermediates.

• MeSH
• biologické modely MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• elongace genetické transkripce MeSH
• fyziologický stres genetika   MeSH
• genetická transkripce * MeSH
• HEK293 buňky MeSH
• helikasy RecQ metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• multienzymové komplexy metabolismus   MeSH
• otevřené čtecí rámce genetika   MeSH
• prekurzory RNA genetika   MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• protein BRCA1 metabolismus   MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikace DNA * MeSH
• ribozomální DNA metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• DNA synthesome MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• DNA-dependentní DNA-polymerasy MeSH
• helikasy RecQ MeSH
• multienzymové komplexy MeSH
• prekurzory RNA MeSH
• proliferační antigen buněčného jádra MeSH
• protein BRCA1 MeSH
• RAD18 protein, human MeSH Prohlížeč
• RECQL5 protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• ribozomální DNA MeSH
• ubikvitinligasy MeSH

R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co-transcriptionally in the opposite orientation to replication fork progression. A recent study has shown that replication forks stalled by co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by ELL-dependent reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds R-loops in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4-ELL pathway for resolution of R-loop-mediated transcription-replication conflicts, which may be involved in R-loop unwinding.

• MeSH
• DEAD-box RNA-helikasy genetika   metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• DNA metabolismus   MeSH
• endonukleasy metabolismus   MeSH
• lidé MeSH
• R-smyčka * MeSH
• replikace DNA * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDX17 protein, human MeSH Prohlížeč
• DEAD-box RNA-helikasy MeSH
• DNA-helikasy MeSH
• DNA MeSH
• endonukleasy MeSH

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adapter ligation, to comprehensively interrogate the human transcriptome at single-molecule and -nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on deadenylation or decapping. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome while inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 rescues RNA length. Our findings reveal RNA decay as a key component of RNA metabolism upon cellular stress that is dependent on stress granule formation.

• Klíčová slova
• RNA decay, cell biology, cell line, genetics, genomics, human, mouse, stress response,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   genetika   MeSH
• DNA-helikasy metabolismus   genetika   MeSH
• exoribonukleasy * metabolismus   genetika   MeSH
• fyziologický stres * genetika   MeSH
• lidé MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny vázající poly-ADP-ribosu * metabolismus   genetika   MeSH
• proteiny vázající RNA MeSH
• ribozomy metabolismus   MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• RRM proteiny * metabolismus   genetika   MeSH
• sekvenční analýza RNA * MeSH
• stabilita RNA * genetika   MeSH
• stresová tělíska metabolismus   genetika   MeSH
• transkriptom MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• DNA-helikasy MeSH
• exoribonukleasy * MeSH
• G3BP1 protein, human MeSH Prohlížeč
• G3BP2 protein, human MeSH Prohlížeč
• proteiny asociované s mikrotubuly MeSH
• proteiny vázající poly-ADP-ribosu * MeSH
• proteiny vázající RNA MeSH
• RNA-helikasy MeSH
• RRM proteiny * MeSH
• XRN1 protein, human MeSH Prohlížeč