RNA Degradosome Research Articles

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

The Sole DEAD-Box RNA Helicase of the Gastric Pathogen Helicobacter pylori Is Essential for Colonization.

ABSTRACTPresent in every kingdom of life, generally in multiple copies, DEAD-box RNA helicases are specialized enzymes that unwind RNA secondary structures. They play major roles in mRNA decay, ribosome biogenesis, and adaptation to cold temperatures. Most bacteria have multiple DEAD-box helicases that present both specialized and partially redundant functions. By using phylogenomics, we revealed that the Helicobacter genus, including the major gastric pathogen H. pylori, is among the exceptions, as it encodes a sole DEAD-box RNA helicase. In H. pylori, this helicase, designated RhpA, forms a minimal RNA degradosome together with the essential RNase, RNase J, a major player in the control of RNA decay. Here, we used H. pylori as a model organism with a sole DEAD-box helicase and investigated the role of this helicase in H. pylori physiology, ribosome assembly, and during in vivo colonization. Our data showed that RhpA is dispensable for growth at 37°C but crucial at 33°C, suggesting an essential role of the helicase in cold adaptation. Moreover, we found that a ΔrhpA mutant was impaired in motility and deficient in colonization of the mouse model. RhpA is involved in the maturation of 16S rRNA at 37°C and is associated with translating ribosomes. At 33°C, RhpA is, in addition, recruited to individual ribosomal subunits. Finally, via its role in the RNA degradosome, RhpA directs the regulation of the expression of its partner, RNase J. RhpA is thus a multifunctional enzyme that, in H. pylori, plays a central role in gene regulation and in the control of virulence.

Structural analysis of mtEXO mitochondrial RNA degradosome reveals tight coupling of nuclease and helicase components

Nuclease and helicase activities play pivotal roles in various aspects of RNA processing and degradation. These two activities are often present in multi-subunit complexes from nucleic acid metabolism. In the mitochondrial exoribonuclease complex (mtEXO) both enzymatic activities are tightly coupled making it an excellent minimal system to study helicase–exoribonuclease coordination. mtEXO is composed of Dss1 3′-to-5′ exoribonuclease and Suv3 helicase. It is the master regulator of mitochondrial gene expression in yeast. Here, we present the structure of mtEXO and a description of its mechanism of action. The crystal structure of Dss1 reveals domains that are responsible for interactions with Suv3. Importantly, these interactions are compatible with the conformational changes of Suv3 domains during the helicase cycle. We demonstrate that mtEXO is an intimate complex which forms an RNA-binding channel spanning its entire structure, with Suv3 helicase feeding the 3′ end of the RNA toward the active site of Dss1.

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome–comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing.

Escherichia coli responds to environmental changes using enolasic degradosomes and stabilized DicF sRNA to alter cellular morphology

Escherichia coli RNase E is an essential enzyme that forms multicomponent ribonucleolytic complexes known as "RNA degradosomes." These complexes consist of four major components: RNase E, PNPase, RhlB RNA helicase, and enolase. However, the role of enolase in the RNase E/degradosome is not understood. Here, we report that presence of enolase in the RNase E/degradosome under anaerobic conditions regulates cell morphology, resulting in Ecoli MG1655 cell filamentation. Under anaerobic conditions, enolase bound to the RNase E/degradosome stabilizes the small RNA (sRNA) DicF, i.e., the inhibitor of the cell division gene ftsZ, through chaperon protein Hfq-dependent regulation. RNase E/enolase distribution changes from membrane-associated patterns under aerobic to diffuse patterns under anaerobic conditions. When the enolase-RNase E/degradosome interaction is disrupted, the anaerobically induced characteristics disappear. We provide a mechanism by which Ecoli uses enolase-bound degradosomes to switch from rod-shaped to filamentous form in response to anaerobiosis by regulating RNase E subcellular distribution, RNase E enzymatic activity, and the stability of the sRNA DicF required for the filamentous transition. In contrast to Ecoli nonpathogenic strains, pathogenic Ecoli strains predominantly have multiple copies of sRNA DicF in their genomes, with cell filamentation previously being linked to bacterial pathogenesis. Our data suggest a mechanism for bacterial cell filamentation during infection under anaerobic conditions.

Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus.

ABSTRACTIn diverse bacterial lineages, multienzyme assemblies have evolved that are central elements of RNA metabolism and RNA-mediated regulation. The aquatic Gram-negative bacterium Caulobacter crescentus, which has been a model system for studying the bacterial cell cycle, has an RNA degradosome assembly that is formed by the endoribonuclease RNase E and includes the DEAD-box RNA helicase RhlB. Immunoprecipitations of extracts from cells expressing an epitope-tagged RNase E reveal that RhlE, another member of the DEAD-box helicase family, associates with the degradosome at temperatures below those optimum for growth. Phenotype analyses of rhlE, rhlB, and rhlE rhlB mutant strains show that RhlE is important for cell fitness at low temperature and its role may not be substituted by RhlB. Transcriptional and translational fusions of rhlE to the lacZ reporter gene and immunoblot analysis of an epitope-tagged RhlE indicate that its expression is induced upon temperature decrease, mainly through posttranscriptional regulation. RNase E pulldown assays show that other proteins, including the transcription termination factor Rho, a second DEAD-box RNA helicase, and ribosomal protein S1, also associate with the degradosome at low temperature. The results suggest that the RNA degradosome assembly can be remodeled with environmental change to alter its repertoire of helicases and other accessory proteins.IMPORTANCE DEAD-box RNA helicases are often present in the RNA degradosome complex, helping unwind secondary structures to facilitate degradation. Caulobacter crescentus is an interesting organism to investigate degradosome remodeling with change in temperature, because it thrives in freshwater bodies and withstands low temperature. In this study, we show that at low temperature, the cold-induced DEAD-box RNA helicase RhlE is recruited to the RNA degradosome, along with other helicases and the Rho protein. RhlE is essential for bacterial fitness at low temperature, and its function may not be complemented by RhlB, although RhlE is able to complement for rhlB loss. These results suggest that RhlE has a specific role in the degradosome at low temperature, potentially improving adaptation to this condition.

The Structure and Function of the Gram-Positive Bacterial RNA Degradosome.

The RNA degradosome is a highly structured protein complex responsible for bulk RNA decay in bacteria. The main components of the complex, ribonucleases, an RNA helicase, and glycolytic enzymes are well-conserved in bacteria. Some components of the degradosome are essential for growth and the disruption of degradosome formation causes slower growth, indicating that this complex is required for proper cellular function. The study of the Escherichia coli degradosome has been performed extensively for the last several decades and has revealed detailed information on its structure and function. On the contrary, the Gram-positive bacterial degradosome, which contains ribonucleases different from the E. coli one, has been studied only recently. Studies on the Gram-positive degradosome revealed that its major component RNase Y was necessary for the full virulence of medically important Gram-positive bacterial pathogens, suggesting that it could be a target of antimicrobial therapy. This review describes the structures and function of Gram-positive bacterial RNA degradosomes, especially those of a Gram-positive model organism Bacillus subtilis, and two important Gram-positive pathogens, Staphylococcus aureus and Streptococcus pyogenes.

Pseudomonas aeruginosa Enolase Influences Bacterial Tolerance to Oxidative Stresses and Virulence.

Pseudomonas aeruginosa is a Gram negative opportunistic pathogenic bacterium, which causes acute and chronic infections. Upon entering the host, bacteria alter global gene expression to adapt to host environment and avoid clearance by the host. Enolase is a glycolytic enzyme involved in carbon metabolism. It is also a component of RNA degradosome, which is involved in RNA processing and gene regulation. Here, we report that enolase is required for the virulence of P. aeruginosa in a murine acute pneumonia model. Mutation of enolase coding gene (eno) increased bacterial susceptibility to neutrophil mediated killing, which is due to reduced tolerance to oxidative stress. Catalases and alkyl hydroperoxide reductases play a major role in protecting the cell from oxidative damages. In the eno mutant, the expression levels of catalases (KatA and KatB) were similar as those in the wild type strain in the presence of H2O2, however, the expression levels of alkyl hydroperoxide reductases (AhpB and AhpC) were significantly reduced. Overexpression of ahpB but not ahpC in the eno mutant fully restored the bacterial resistance to H2O2 as well as neutrophil mediated killing, and partially restored bacterial virulence in the murine acute pneumonia model. Therefore, we have identified a novel role of enolase in the virulence of P. aeruginosa.

Localization of Components of the RNA-Degrading Machine in Bacillus subtilis.

In bacteria, the control of mRNA stability is crucial to allow rapid adaptation to changing conditions. In most bacteria, RNA degradation is catalyzed by the RNA degradosome, a protein complex composed of endo- and exoribonucleases, RNA helicases, and accessory proteins. In the Gram-positive model organism Bacillus subtilis, the existence of a RNA degradosome assembled around the membrane-bound endoribonuclease RNase Y has been proposed. Here, we have studied the intracellular localization of the protein that have been implicated in the potential B. subtilis RNA degradosome, i.e., polynucleotide phosphorylase, the exoribonucleases J1 and J2, the DEAD-box RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase. Our data suggests that the bulk of these enzymes is located in the cytoplasm. The RNases J1 and J2 as well as the RNA helicase CshA were mainly localized in the peripheral regions of the cell where also the bulk of messenger RNA is localized. We were able to demonstrate active exclusion of these proteins from the transcribing nucleoid. Taken together, our findings suggest that the interactions of the enzymes involved in RNA degradation in B. subtilis are rather transient.

Structural elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA degradosome.

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 Å crystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells.

Cyanobacterial RNA Helicase CrhR Localizes to the Thylakoid Membrane Region and Cosediments with Degradosome and Polysome Complexes in Synechocystis sp. Strain PCC 6803.

The cyanobacterium Synechocystis sp. strain PCC 6803 encodes a single DEAD box RNA helicase, CrhR, whose expression is tightly autoregulated in response to cold stress. Subcellular localization and proteomic analysis results indicate that CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with polysome and RNA degradosome components. Evidence is presented that either functional RNA helicase activity or a C-terminal localization signal was required for polysome but not thylakoid membrane localization. Polysome fractionation and runoff translation analysis results indicate that CrhR associates with actively translating polysomes. The data implicate a role for CrhR in translation or RNA degradation in the thylakoid region related to thylakoid biogenesis or stability, a role that is enhanced at low temperature. Furthermore, CrhR cosedimentation with polysome and RNA degradosome complexes links alteration of RNA secondary structure with a potential translation-RNA degradation complex in Synechocystis The interaction between mRNA translation and degradation is a major determinant controlling gene expression. Regulation of RNA function by alteration of secondary structure by RNA helicases performs crucial roles, not only in both of these processes but also in all aspects of RNA metabolism. Here, we provide evidence that the cyanobacterial RNA helicase CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with actively translating polysomes and RNA degradosome components. These findings link RNA helicase alteration of RNA secondary structure with translation and RNA degradation in prokaryotic systems and contribute to the data supporting the idea of the existence of a macromolecular machine catalyzing these reactions in prokaryotic systems, an association hitherto recognized only in archaea and eukarya.

Spatial organization shapes the turnover of a bacterial transcriptome.

Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Time-resolved RNA-sequencing revealed that degradation rates of inner-membrane-protein mRNAs are on average greater that those of the other mRNAs and that this selective destabilization of inner-membrane-protein mRNAs is abolished by dissociating the RNA degradosome from the membrane. Together, these results demonstrate that the bacterial transcriptome is spatially organized and suggest that this organization shapes the post-transcriptional dynamics of mRNAs.

Three-Dimensional Super-Resolution Imaging of the RNA Degradation Machinery in Caulobacter Crescentus

Due the small size of bacteria and the diffraction limit (DL), obtaining precise information on the location of biomolecules through the use of conventional fluorescence imaging is difficult. However, the conventional resolution of optical microscopes (∼200 nm) can be circumvented by the time-sequential super-localization of single-molecule fluorescent labels, resulting in ∼5x resolution enhancement. We applied these super-resolution techniques, in combination with a double-helix point spread function microscope, to study the three-dimensional (3D) spatial organization of the RNA degradation machinery in Caulobacter crescentus (C. crescentus). The RNA degradosome plays a dominant role in mRNA degradation, a key step in gene expression. RNaseE is a key component in the RNA degradosome, and is thought to be the scaffold for this complex in C. crescentus. Previous diffraction-limited fluorescence imaging has shown that C. crescentus RNaseE exhibits a patchy localization throughout the nucleoid. We report the first 3D super-resolution images of RNaseE in live C. crescentus, with careful attention to remove any spurious clustering from over-counting of blinking fluorescent protein labels. We use the acquired localizations to quantify RNaseE's spatial distribution and find a non-random, clearly clustered distribution of this protein. Moreover, most RNaseE molecules are localized in regular clusters spaced along the cell axis, and it is possible to extract specific measures of cluster size and other parameters. Disturbing transcription perturbs the protein distribution, resulting in more diffuse RNaseE localization. Our results suggest that mRNA decay is spatially organized within C. crescentus, and that this organization is related to the function of RNaseE in mRNA homeostasis.

The Protein Interaction of RNA Helicase B (RhlB) and Polynucleotide Phosphorylase (PNPase) Contributes to the Homeostatic Control of Cysteine in Escherichia coli

PNPase, one of the major enzymes with 3′ to 5′ single-stranded RNA degradation and processing activities, can interact with the RNA helicase RhlB independently of RNA degradosome formation in Escherichia coli. Here, we report that loss of interaction between RhlB and PNPase impacts cysteine homeostasis in E. coli. By random mutagenesis, we identified a mutant RhlBP238L that loses 75% of its ability to interact with PNPase but retains normal interaction with RNase E and RNA, in addition to exhibiting normal helicase activity. Applying microarray analyses to an E. coli strain with impaired RNA degradosome formation, we investigated the biological consequences of a weakened interaction between RhlB and PNPase. We found significant increases in 11 of 14 genes involved in cysteine biosynthesis. Subsequent Northern blot analyses showed that the up-regulated transcripts were the result of stabilization of the cysB transcript encoding a transcriptional activator for the cys operons. Furthermore, Northern blots of PNPase or RhlB mutants showed that RhlB-PNPase plays both a catalytic and structural role in regulating cysB degradation. Cells expressing the RhlBP238L mutant exhibited an increase in intracellular cysteine and an enhanced anti-oxidative response. Collectively, this study suggests a mechanism by which bacteria use the PNPase-RhlB exosome-like complex to combat oxidative stress by modulating cysB mRNA degradation.

DsrA regulatory RNA represses both hns and rbsD mRNAs through distinct mechanisms in Escherichia coli.

The 87 nucleotide long DsrA sRNA has been mostly studied for its translational activation of the transcriptional regulator RpoS. However, it also represses hns mRNA, which encodes H-NS, a major regulator that affects expression of nearly 5% of Escherichia coli genes. A speculative model previously suggested that DsrA would block hns mRNA translation by binding simultaneously to start and stop codon regions of hns mRNA (coaxial model). Here, we show that DsrA efficiently blocked translation of hns mRNA by base-pairing immediately downstream of the start codon. In addition, DsrA induced hns mRNA degradation by actively recruiting the RNA degradosome complex. Data presented here led to a model of DsrA action on hns mRNA, which supports a canonical mechanism of sRNA-induced mRNA degradation by binding to the translation initiation region. Furthermore, using MS2-affinity purification coupled with RNA sequencing technology (MAPS), we also demonstrated that DsrA targets rbsD mRNA, involved in ribose utilization. Surprisingly, DsrA base pairs far downstream of rbsD start codon and induces rapid degradation of the transcript. Thus, our study enables us to draw an extended DsrA targetome.

Experimental evolution of enhanced growth by Bacillus subtilis at low atmospheric pressure: genomic changes revealed by whole-genome sequencing.

Knowledge of how microorganisms respond and adapt to low-pressure (LP) environments is limited. Previously, Bacillus subtilis strain WN624 was grown at the near-inhibitory LP of 5 kPa for 1,000 generations and strain WN1106, which exhibited increased relative fitness at 5 kPa, was isolated. Genomic sequence differences between ancestral strain WN624 and LP-evolved strain WN1106 were identified using whole-genome sequencing. LP-evolved strain WN1106 carried amino acid-altering mutations in the coding sequences of only seven genes (fliI, parC, ytoI, bacD, resD, walK, and yvlD) and a single 9-nucleotide in-frame deletion in the rnjB gene that encodes RNase J2, a component of the RNA degradosome. By using a collection of frozen stocks of the LP-evolved culture taken at 50-generation intervals, it was determined that (i) the fitness increase at LP occurred rapidly, while (ii) mutation acquisition exhibited complex kinetics. A knockout mutant of rnjB was shown to increase the competitive fitness of B. subtilis at both LP and standard atmospheric pressure.

RNA degradosomes in bacteria and chloroplasts: classification, distribution and evolution of RNase E homologs.

Ribonuclease E (RNase E) of Escherichia coli, which is the founding member of a widespread family of proteins in bacteria and chloroplasts, is a fascinating enzyme that still has not revealed all its secrets. RNase E is an essential single-strand specific endoribonuclease that is involved in the processing and degradation of nearly every transcript in E. coli. A striking enzymatic property is a preference for substrates with a 5' monophosphate end although recent work explains how RNase E can overcome the protection afforded by the 5' triphosphate end of a primary transcript. Other features of E. coli RNase E include its interaction with enzymes involved in RNA degradation to form the multienzyme RNA degradosome and its localization to the inner cytoplasmic membrane. The N-terminal catalytic core of the RNase E protomer associates to form a tetrameric holoenzyme. Each RNase E protomer has a large C-terminal intrinsically disordered (ID) noncatalytic region that contains sites for interactions with protein components of the RNA degradosome as well as RNA and phospholipid bilayers. In this review, RNase E homologs have been classified into five types based on their primary structure. A recent analysis has shown that type I RNase E in the γ-proteobacteria forms an orthologous group of proteins that has been inherited vertically. The RNase E catalytic core and a large ID noncatalytic region containing an RNA binding motif and a membrane targeting sequence are universally conserved features of these orthologs. Although the ID noncatalytic region has low composition and sequence complexity, it is possible to map microdomains, which are short linear motifs that are sites of interaction with protein and other ligands. Throughout bacteria, the composition of the multienzyme RNA degradosome varies with species, but interactions with exoribonucleases (PNPase, RNase R), glycolytic enzymes (enolase, aconitase) and RNA helicases (DEAD-box proteins, Rho) are common. Plasticity in RNA degradosome composition is due to rapid evolution of RNase E microdomains. Characterization of the RNase E-PNPase interaction in α-proteobacteria, γ-proteobacteria and cyanobacteria suggests that it arose independently several times during evolution, thus conferring an advantage in control and coordination of RNA processing and degradation.

A career in (mRNA) decay.

A career in (mRNA) decay.

Way to go, RNA.

Way to go, RNA.

Membrane recognition and dynamics of the RNA degradosome.

RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies.