RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori
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PROVIDER: PRJEB42684 | ENA |
REPOSITORIES: ENA
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Project description:Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3’-5’ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The "prototypical" Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, that include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.
2021-04-08 | PXD023837 | Pride
Project description:Ribonucleases (RNases) are central actors in post-transcriptional regulation, a major level of regulation of gene expression in all cells. This control plays an important role in the bacterial pathogen Helicobacter pylori, although only the function of RNase J was characterized so far. Here, we studied the RNase R enzyme from H. pylori, a 3’-5’ exoribonuclease whose ortholog in Escherichia coli was reported to display, in addition, helicase activity and to be able to hydrolyze RNA substrates with double stranded structures. We observed that HpRNase R protein does not carry the domains responsible for helicase activity in E. coli and accordingly that the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The RNase R helicase domain loss is widespread among the Campylobacterota and occurred gradually during their evolution. Furthermore, an in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori, was discovered. Phylogenomics suggests that this interaction might occur in other bacteria of the phylum Campylobacterota. Purified RhpA facilitates the degradation of double stranded RNA substrates by HpRNase R, showing that this complex is functional. HpRNase R has a minor role of in 5S rRNA maturation and, as shown by RNA-Seq, few targets in H. pylori all of them being included in the RhpA regulon. In conclusion, we describe a new type of RNase R that lacks some of the features that were considered as hallmarks of RNase R proteins, but that has co-opted another RNA helicase, which we hypothesize helps it accomplish some of its functions in vivo.
2021-06-19 | E-MTAB-10045 | biostudies-arrayexpress
Project description:In Helicobacter pylori, the major actor in post-transcriptional regulation is an RNA degradosome assembly composed of the essential ribonuclease RNase J and the DEAD-box RNA helicase RhpA. Here, we describe the oligomeric state and post-translational modifications of this protein complex. Our crystal structure of RNase J at 2.75 Å resolution reveals a homotetrameric quaternary state, and solution studies show that purified RNase J forms monomers and dimers, that RhpA is a monomer, and that the two proteins interact to form a stable complex with a 1:1 stoichiometry. Using mass spectrometry, we observed that RNase J is acetylated on multiple residues, one of which, K649, is important for RNase J oligomerization. Mutations targeting this residue and others affect the dimerization and in vitro activity of RNase J, suggesting that the dimer is the active form. In H. pylori, we observed that several of the identified acetylated residues impact on cell morphology, suggesting their importance for RNase J cellular function. Using H. pylori mutants, we found that several pathways contribute to RNase J acetylation. We propose acetylation as a regulatory level controlling RNase J properties and as a consequence the activity of the H. pylori RNA degradosome.
2023-12-12 | PXD027670 | Pride
Project description:Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade and plays both RING (really interesting new gene)-dependent and RING-independent roles in RIG-I-mediated IFN induction. We report that the PRY-SPRY domain of TRIM25 interacts with the N-terminal extension (NTE) of DEAD-box helicase 3X (DDX3X), a host protein with multiple roles in RLR signalling. Gene reporter assays and knockdown studies reveal DDX3X and TRIM25 cooperate to activate the IFN- promoter following RIG-I activation independent of DDX3X’s catalytic activity. We also show that TRIM25 ubiquitinates DDX3X at several lysine residues in vitro and in cells.
2021-09-08 | PXD028122 | Pride
Project description:Organ-differential Regulation of Vascular Development by DEAD-box Helicase 24
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Project description:How cells regulate gene expression in a precise spatiotemporal manner during organismal development is a fundamental question in biology. Although the role of transcriptional condensates in gene regulation has been established, little is known about the function and regulation of these molecular assemblies in the context of animal development and physiology. Here we show that the evolutionarily conserved DEAD-box helicase DDX-23 controls cell fate in Caenorhabditis elegans by binding to and facilitating the condensation of MAB-10, the Caenorhabditis elegans homolog of mammalian NGFI-A-binding (NAB) protein. MAB-10 is a transcriptional cofactor that functions with the early growth response (EGR) protein LIN-29 to regulate the transcription of genes required for exiting the cell cycle, terminal differentiation, and the larval-to-adult transition. We suggest that DEAD-box helicase proteins function more generally during animal development to control the condensation of NAB proteins important in cell identity and that this mechanism is evolutionarily conserved. In mammals, such a mechanism might underlie terminal cell differentiation and when misregulated might promote cancerous growth.
2023-05-28 | GSE151399 | GEO
Project description:Although DEAD-box helicase 41 (DDX41) is implicated in oncogenic and innate immune mechanisms, there are many unanswered questions about how the ever-increasing spectrum of genetic variants impacts DDX41 activity. Here, we describe a facile genetic rescue assay that discriminates activities of DDX41 from those of human myeloid malignancy-linked germline and somatic DDX41 mutants. Our analyses revealed that the variants were impaired in their intrinsic RNA-regulatory activities and to induce monocytic differentiation markers. It will be instructive to extend these analyses to more broadly conduct structure/function assessments for clinical genetic curation and leverage the quantitative assay to elucidate mechanisms and interventions that promote and/or oppose DDX41 function, thereby influencing DDX41-linked pathogenicity.
2022-12-04 | GSE208400 | GEO
Project description:Accurate gene expression requires the coordination of RNA processing with assembly of messenger RNA-protein (mRNP) complex. RNA helicases are a class of enzymes that unwind RNA duplexes in vitro and have been are proposed to remodel RNA structure in vivo. Herein, we provide evidence that the DEAD-box protein Dbp2 remodels RNA structure to facilitate efficient pre-mRNA processing in S. cerevisiae. First, we find that Dbp2 associates with the 3’ ends and 3’ splice-sites of mRNAs genome-wide. Using structure-seq to map RNA secondary structure, we find altered secondary structures in dbp2∆ cells that overlap polyadenylation elements and correlate with inefficient termination. We also identify a role for Dbp2 in pre-mRNA splicing and show that both splicing and termination require Dbp2 helicase activity. This reveals that DEAD-box RNA helicases unwind structure in vivo and that structural alteration of pre-mRNA is essential for proper gene expression.
2019-05-01 | GSE106477 | GEO
Project description:Accurate gene expression requires the coordination of RNA processing with assembly of messenger RNA-protein (mRNP) complex. RNA helicases are a class of enzymes that unwind RNA duplexes in vitro and have been are proposed to remodel RNA structure in vivo. Herein, we provide evidence that the DEAD-box protein Dbp2 remodels RNA structure to facilitate efficient pre-mRNA processing in S. cerevisiae. First, we find that Dbp2 associates with the 3’ ends and 3’ splice-sites of mRNAs genome-wide. Using structure-seq to map RNA secondary structure, we find altered secondary structures in dbp2∆ cells that overlap polyadenylation elements and correlate with inefficient termination. We also identify a role for Dbp2 in pre-mRNA splicing and show that both splicing and termination require Dbp2 helicase activity. This reveals that DEAD-box RNA helicases unwind structure in vivo and that structural alteration of pre-mRNA is essential for proper gene expression.
2019-05-01 | GSE106478 | GEO
Project description:RNA interference (RNAi) is a potent mechanism for down-regulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary siRNAs. Exogenous double stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear if the sub-cellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved FG domain containing DEAD-box helicase, localizes in P-granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA targeted mRNA in distinct cytoplasmic compartments. Examination of exogenous dsRNA trigger derived siRNA in wildtype and rde-12 mutant animals
2014-03-27 | E-GEOD-53830 | biostudies-arrayexpress