The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.     

New insights into the cellular organization of the RNA processing and degradation machinery of Escherichia coli

Ribonuclease E (RNase E) is a component of the Escherichia coli RNA degradosome, a multiprotein complex that also includes RNA helicase B (RhlB), polynucleotide phosphorylase (PNPase) and enolase. The degradosome plays a key role in RNA processing and degradation. The degradosomal proteins are organized as a cytoskeletal-like structure within the cell that has been thought to be associated with the cytoplasmic membrane. The article by Khemici et al. in the current issue of Molecular Microbiology reports that RNase E can directly interact with membrane phospholipids in vitro. The RNase E-membrane interaction is likely to play an important role in the membrane association of the degradosome system. These findings shed light on important but largely unexplored aspects of cellular structure and function, including the organization of the RNA processing machinery of the cell and of bacterial cytoskeletal elements in general.     

DEAD box RhlB RNA helicase physically associates with exoribonuclease PNPase to degrade double-stranded RNA independent of the degradosome-assembling region of RNase E

The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.     

Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.     

Self-Assembly of the Bacterial Cytoskeleton-Associated RNA Helicase B Protein into Polymeric Filamentous Structures

The Escherichia coli RNA degradosome proteins are organized into a helical cytoskeletal-like structure within the cell. Here we describe the ATP-dependent assembly of the RhlB component of the degradosome into polymeric filamentous structures in vitro, which suggests that extended polymers of RhlB are likely to comprise a basic core element of the degradosome cytoskeletal structures.The RNA degradosome plays an essential role in normal RNA processing and degradation. Within the cell, the degradosome proteins (RNA helicase B [RhlB], RNase E, polynucleotide phosphorylase [PNPase], and enolase) (4, 13, 15, 16) are organized into coiled structures that resemble the pole-to-pole helical structures of the MreB and MinCDE bacterial cytoskeletal systems (4, 12, 13). However, the degradosomal structures are also present in cells that lack the MreB and MinCDE cytoskeletal elements, suggesting that the degradosomal structures may be part of an independent class of prokaryotic cytoskeletal elements (19-21).One of the degradosomal proteins, RhlB, is organized into similar helical cellular structures in cells that lack the other degradosome proteins (Fig. ​(Fig.11 A). In addition, RhlB recruits PNPase to the helical framework in the absence of other degradosome proteins, suggesting that the RhlB structures are core elements of the degradosomal cytoskeletal-like elements of the cell (Fig. ​(Fig.1B)1B) (20). The cellular RhlB structures could be generated in two ways: (i) individual RhlB molecules may bind to an as-yet-undefined underlying track, or (ii) RhlB may polymerize to form the filamentous helical structures independent of any underlying template.Open in a separate windowFIG. 1.The RhlB filamentous cytoskeletal-like structures. (A) Cellular organization of RhlB based on immunofluorescence microscopy using purified anti-RhlB antibody in the absence of RNase E filamentous elements in AT8 cells (rne^1-417), which fail to generate RNase E coiled structures because of the absence of the RNase E cytoskeletal localization domain (20). (B) Proposed model for the cytoskeletal-like organization of the RNA degradosome (modified from reference 20). Arcs depict the RNase E (blue) and RhlB (red) helical strands. It is not known whether the RNase E helical strand is formed by RNase E polymerization or by the association of RNase E with an unknown underlying cytoskeletal structure. Enolase (Eno) and PNPase are shown in gray. Molecular dimensions and stoichiometry of the proteins were arbitrarily chosen to simplify the figure. (C to H) Electron micrographs of uranyl acetate-stained RhlB filaments (C, E, and H) and RhlB sheets (D). Unless otherwise indicated, the sample contained 9 μM RhlB, 2 mM ATP, 5 mM MgCl₂, and 5 mM CaCl₂. (E) Calcium was omitted. (F) ATP was omitted. (G and H) ATP was replaced by ATPγS (G) or AMP-PNP (H). Samples were loaded on glow-discharged 300-mesh carbon-coated copper grids and then stained. Images were taken with a JEOL 100CX transmission electron microscope. Magnification, ×10,000 to 50,000.Here we report that RhlB can self-assemble into extended polymeric structures in vitro in a process that requires ATP binding but not ATP hydrolysis. It is likely that extended RhlB polymers such as those described here are the basic components of the RhlB filamentous helical elements that comprise the core of the degradosomal cytoskeletal structures of the Escherichia coli cell.Evidence that RhlB can self-assemble into filamentous polymeric structures came from electron microscopic studies of purified His-tagged RhlB negatively stained with 2% uranyl acetate. This staining showed large numbers of long uniform filamentous structures when the purified protein was incubated in the presence of ATP and Ca²⁺ (Fig. ​(Fig.1C).1C). The filaments were 25 ± 1.8 nm wide (n = 91; mean ± standard deviation) and were generally more than 10 μm long. Some wider sheets were also observed (Fig. ​(Fig.1D).1D). Optimal assembly of the RhlB filamentous structures required ATP and Ca²⁺, as shown by the observation that only occasional single structures were present when the polymerization reaction was carried out in the absence of Ca²⁺ (Fig. ​(Fig.1E)1E) or ATP (Fig. ​(Fig.1F).1F). The polymeric RhlB-His structures were observed with approximately similar frequencies when ATP was replaced by the nonhydrolyzable ATP analog adenosine 5′-(γ-thiotriphosphate) (ATPγS) or AMP-PNP (Fig. 1G and H).Cellular localization studies showed that the presence of the His tag did not interfere with the ability of RhlB to form the helical cellular structures. Thus, RhlB-His was present in extended helical filamentous structures that were indistinguishable from those formed by untagged RhlB (20, 21). Similarly, the RhlB-His structures recruited PNPase to the helical framework in a manner similar to untagged RhlB (20) (see Fig. S1 in the supplemental material).Immunogold staining showed that the filaments and sheets were decorated with gold particles when stained with mouse anti-His tag antibody and gold-labeled secondary antibody (Fig. ​(Fig.22 A to C), confirming that the structures were composed of RhlB. In contrast, the structures were not decorated with gold particles in the absence of the primary antibody or when mouse anti-His tag antibody was replaced by nonimmune mouse IgG (Fig. ​(Fig.2D2D and E). The polymeric structures were observed with C-terminally His-tagged RhlB, which is functional in terms of helicase activity (8), but not when the tag was present at the amino terminus of the protein, where the His tag may interfere with RhlB self-assembly.Open in a separate windowFIG. 2.Immunogold electron microscopy of RhlB structures. Samples were prepared as describe for Fig. ​Fig.1C,1C, except that the grids were stained with 2% uranyl acetate after exposure to primary and/or secondary antibodies as indicated. (A to C) RhlB structures decorated with 10-nm gold particles in samples stained with mouse anti-His tag monoclonal antibody and gold-labeled secondary antibody. (A) single filaments; (B) clustered filaments; (C) a single RhlB filament and RhlB sheet. Arrows indicate gold particles. (D) The primary mouse anti-His tag antibody was replaced by mouse IgG. (E) The primary antibody was omitted.Changes in light scattering were used to follow the course of polymerization and to compare polymerization conditions in a more quantitative way than is possible by electron microscopy. The initial rate of increase in scattering was used to estimate polymerization rate (see Table S1 in the supplemental material). Significant rates of polymerization were observed in the presence of ATP and Ca²⁺, whereas there was very little increase in light scattering in the absence of nucleotide and/or Ca²⁺ (Fig. ​(Fig.33 A). ADP was less effective than ATP, whereas AMP and cyclic AMP (cAMP) were inactive (Fig. ​(Fig.3B).3B). In the presence of Ca²⁺, the extent and rate of RhlB polymerization varied as a function of ATP concentration (Fig. ​(Fig.3C).3C). Millimolar concentrations of Ca²⁺ were required to produce a measurable rate of polymerization in the light scattering assay (Fig. ​(Fig.3A).3A). It is not known how these relatively high concentrations of Ca²⁺ promote the in vitro polymerization of RhlB and other cytoskeletal proteins, such as MreB and FtsZ (1, 11, 12, 14, 23).Open in a separate windowFIG. 3.RhlB polymerization. (A and B) RhlB polymerization as shown by 90° light scattering is indicated in arbitrary units (a.u.). RhlB polymerization was followed at room temperature in a 1-cm light path quartz cuvette using a Hitachi fluorometer (FL-2500) set to 400 V with excitation and emission wavelengths set at 455 nm and a slit width of 10 nm. The reaction (100 μl volume) was performed in a polymerization buffer (50 mM Tris, 50 mM KCl, 5 mM MgCl₂; pH 8) as indicated. (A) The sample contained 9 μM RhlB-His, 1 mM ATP, and either 10 mM CaCl₂, 7.5 mM CaCl₂, or no CaCl₂ (squares). In the lower three curves the samples lacked ATP or CaCl₂, as indicated. (B) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and 1 mM adenosine nucleotides: ATP, ADP, AMP, cAMP, AMP-PNP, and ATPγS. (C) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and ATP as indicated (1 mM, 0.75 mM, 0.5 mM, 0.25 mM, or 0.1 mM ATP). (D) Effects of Ca²⁺ concentration on RhlB sedimentation in the presence of 2 mM ATP. RhlB in the pellet, expressed as a percentage of total RhlB present in the polymerization reaction mixture, was plotted against calcium concentration. The insert shows an example of a Coomassie blue-stained gel of supernatant (S) and pellet (P) fractions from the sedimentation assay in the presence of ATP and Ca²⁺ (see results for ATP in Table ​Table11).The nonhydrolyzable ATP analogs ATPγS and AMP-PNP were approximately equivalent to ATP in promoting polymerization as monitored by the light scattering assay (Fig. ​(Fig.3B)3B) as well as in the electron microscopic studies. This suggests that nucleotide binding, but not hydrolysis, is required to promote RhlB polymerization. In this regard, RhlB resembles a number of other proteins, including F-actin, MreB, and MinD, where polymerization is induced by nucleotide binding (2, 5, 6, 18, 22). In these systems, subsequent ATP hydrolysis induces depolymerization, providing the basis for the dynamic behavior of the polymers within the cell. RhlB is an RNA-dependent ATPase (7), but it is not yet known whether ATP hydrolysis is associated with depolymerization in the RhlB system.Similar results were obtained when the extent of polymerization was monitored by a sedimentation assay, measuring the proportion of RhlB in the pellet fraction after centrifugation at 278,000 × g for 10 min (Table ​(Table1;1; Fig. ​Fig.3D).3D). Essentially all of the protein was sedimentable at pH 8 in the presence of Ca²⁺ and ATP. RhlB sedimentation returned to background levels when EGTA or EDTA was added to the reaction mixture (Table ​(Table1),1), confirming the Ca²⁺ requirement for RhlB polymerization in the electron microscopic and light scattering analyses. ATPγS was equivalent to ATP in the sedimentation assay, confirming the results described above. The relatively high background of RhlB sedimentation was not affected by prespinning the samples prior to addition of nucleotides and/or Ca²⁺.

TABLE 1.

Sedimentation assay for RhlB polymerization

Molecular recognition of RhlB and RNase D in the Caulobacter crescentus RNA degradosome

The endoribonuclease RNase E is a key enzyme in RNA metabolism for many bacterial species. In Escherichia coli, RNase E contributes to the majority of RNA turnover and processing events, and the enzyme has been extensively characterized as the central component of the RNA degradosome assembly. A similar RNA degradosome assembly has been described in the α-proteobacterium Caulobacter crescentus, with the interacting partners of RNase E identified as the Kreb''s cycle enzyme aconitase, a DEAD-box RNA helicase RhlB and the exoribonuclease polynucleotide phosphorylase. Here we report that an additional degradosome component is the essential exoribonuclease RNase D, and its recognition site within RNase E is identified. We show that, unlike its E. coli counterpart, C. crescentus RhlB interacts directly with a segment of the N-terminal catalytic domain of RNase E. The crystal structure of a portion of C. crescentus RNase E encompassing the helicase-binding region is reported. This structure reveals that an inserted segment in the S1 domain adopts an α-helical conformation, despite being predicted to be natively unstructured. We discuss the implications of these findings for the organization and mechanisms of the RNA degradosome.     

Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.     

Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli

The RNA degradosome is a multi-enzyme assembly that contributes to key processes of RNA metabolism, and it engages numerous partners in serving its varied functional roles. Small domains within the assembly recognize collectively a diverse range of macromolecules, including the core protein components, the cytoplasmic lipid membrane, mRNAs, non-coding regulatory RNAs and precursors of structured RNAs. We present evidence that the degradosome can form a stable complex with the 70S ribosome and polysomes, and we demonstrate the proximity in vivo of ribosomal proteins and the scaffold of the degradosome, RNase E. The principal interactions are mapped to two, independent, RNA-binding domains from RNase E. RhlB, the RNA helicase component of the degradosome, also contributes to ribosome binding, and this is favoured through an activating interaction with RNase E. The catalytic activity of RNase E for processing 9S RNA (the ribosomal 5S RNA precursor) is repressed in the presence of the ribosome, whereas there is little affect on the cleavage of single-stranded substrates mediated by non-coding RNA, suggestings that the enzyme retains capacity to cleave unstructured substrates when associated with the ribosome. We propose that polysomes may act as antennae that enhance the rates of capture of the limited number of degradosomes, so that they become recruited to sites of active translation to act on mRNAs as they become exposed or tagged for degradation.     

Recognition and cooperation between the ATP-dependent RNA helicase RhlB and ribonuclease RNase E

The Escherichia coli protein RhlB is an ATP-dependent motor that unfolds structured RNA for destruction by partner ribonucleases. In E. coli, and probably many other related gamma-proteobacteria, RhlB associates with the essential endoribonuclease RNase E as part of the multi-enzyme RNA degradosome assembly. The interaction with RNase E boosts RhlB's ATPase activity by an order of magnitude. Here, we examine the origins and implications of this effect. The location of the interaction sites on both RNase E and RhlB are refined and analysed using limited protease digestion, domain cross-linking and homology modelling. These data indicate that RhlB's carboxy-terminal RecA-like domain engages a segment of RNase E that is no greater than 64 residues. The interaction between RhlB and RNase E has two important consequences: first, the interaction itself stimulates the unwinding and ATPase activities of RhlB; second, RhlB gains proximity to two RNA-binding sites on RNase E, with which it cooperates to unwind RNA. Our homology model identifies a pattern of residues in RhlB that may be key for recognition of RNase E and which may communicate the activating effects. Our data also suggest that the association with RNase E may partially repress the RNA-binding activity of RhlB. This repression may in fact permit the interplay of the helicase and adjacent RNA binding segments as part of a process that steers substrates to either processing or destruction, depending on context, within the RNA degradosome assembly.     

The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex

In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram‐positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein–protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA‐like domains that are found in all DEAD box RNA helicases and a C‐terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C‐terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome.     

The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome

The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonuclease''s degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity.     

Current perspectives of the <Emphasis Type="Italic">Escherichia coli</Emphasis> RNA degradosome

Many biological processes in the cell are linked to RNA metabolism and therefore have implications for a wide range of biotechnological applications. The processing and degradation of RNA plays an important role in RNA metabolism with often the same enzymes being involved in both processes. In this review, we highlight the dynamic nature of the structural components of the Escherichia coli RNA degradosome which is a large multiprotein complex that plays an important role in RNA degradation. The activities of the individual components of the degradosome are also discussed. The RNA degradosome forms part of the bacterial cytoskeleton and associated proteins, such as molecular chaperones, may aid in the compartmentalization of enzymatic activities and cytoskeletal organization. An enhanced understanding of the components of the RNA degradosome in other bacterial species will certainly aid in their evaluation as potential antimicrobial agents.     

The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE

The non-catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome. Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD-box RNA helicase, RhlB. We show that three other DEAD-box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro. Their binding differs from that of RhlB because it is not affected by the R730A mutation. Furthermore, the deletion of residues 791-843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA. Therefore, RNase E has at least two RNA helicase binding sites. Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP-dependent activity that facilitates the degradation of structured RNA by PNPase. Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.     

Analysis of the RNA degradosome complex in Vibrio angustum S14

The RNA degradosome is built on the C-terminal half of ribonuclease E (RNase E) which shows high sequence variation, even amongst closely related species. This is intriguing given its central role in RNA processing and mRNA decay. Previously, we have identified RhlB (ATP-dependent DEAD-box RNA helicase)-binding, PNPase (polynucleotide phosphorylase)-binding and enolase-binding microdomains in the C-terminal half of Vibrio angustum S14 RNase E, and have shown through two-hybrid analysis that the PNPase and enolase-binding microdomains have protein-binding function. We suggest that the RhlB-binding, enolase-binding and PNPase-binding microdomains may be interchangeable between Escherichia coli and V. angustum S14 RNase E. In this study, we used two-hybrid techniques to show that the putative RhlB-binding microdomain can bind RhlB. We then used Blue Native-PAGE, a technique commonly employed in the separation of membrane protein complexes, in a study of the first of its kind to purify and analyse the RNA degradosome. We showed that the V. angustum S14 RNA degradosome comprises at least RNase E, RhlB, enolase and PNPase. Based on the results obtained from sequence analyses, two-hybrid assays, immunoprecipitation experiments and Blue Native-PAGE separation, we present a model for the V. angustum S14 RNA degradosome. We discuss the benefits of using Blue Native-PAGE as a tool to analyse the RNA degradosome, and the implications of microdomain-mediated RNase E interaction specificity.     

Enolase in the RNA degradosome plays a crucial role in the rapid decay of glucose transporter mRNA in the response to phosphosugar stress in Escherichia coli

The ptsG mRNA encoding the major glucose transporter is rapidly degraded in an RNase E-dependent manner in response to the accumulation of glucose 6-P or fructose 6-P when the glycolytic pathway is blocked at its early steps in Escherichia coli. RNase E, a major endonuclease, is associated with polynucleotide phosphorylase (PNPase), RhlB helicase and a glycolytic enzyme, enolase, which bind to its C-terminal scaffold region to form a multienzyme complex called the RNA degradosome. The role of enolase within the RNase E-based degradosome in RNA decay has been totally mysterious. In this article, we demonstrate that the removal of the scaffold region of RNase E suppresses the rapid degradation of ptsG mRNA in response to the metabolic stress without affecting the expression of ptsG mRNA under normal conditions. We also demonstrate that the depletion of enolase but not the disruption of pnp or rhlB eliminates the rapid degradation of ptsG mRNA. Taken together, we conclude that enolase within the degradosome plays a crucial role in the regulation of ptsG mRNA stability in response to a metabolic stress. This is the first instance in which a physiological role for enolase in the RNA degradosome has been demonstrated. In addition, we show that PNPase and RhlB within the degradosome cooperate to eliminate short degradation intermediates of ptsG mRNA.     

The assembly and distribution in vivo of the Escherichia coli RNA degradosome

We report an analysis in vivo of the RNA degradosome assembly of Escherichia coli. Employing fluorescence microscopy imaging and fluorescence energy transfer (FRET) measurements, we present evidence for in vivo pairwise interactions between RNase E–PNPase (polynucleotide phosphorylase), and RNase E–Enolase. These interactions are absent in a mutant strain with genomically encoded RNase E that lacks the C-terminal half, supporting the role of the carboxy-end domain as the scaffold for the degradosome. We also present evidence for in vivo proximity of Enolase–PNPase and Enolase–RhlB. The data support a model for the RNA degradosome (RNAD), in which the RNase E carboxy-end is proximal to PNPase, more distant to Enolase, and more than 10 nm from RhlB helicase. Our measurements were made in strains with mono-copy chromosomal fusions of the RNAD enzymes with fluorescent proteins, allowing measurement of the expression of the different proteins under different growth and stress conditions.     

Potential Regulatory Interactions of Escherichia coli RraA Protein with DEAD-box Helicases

Members of the DEAD-box family of RNA helicases contribute to virtually every aspect of RNA metabolism, in organisms from all domains of life. Many of these helicases are constituents of multicomponent assemblies, and their interactions with partner proteins within the complexes underpin their activities and biological function. In Escherichia coli the DEAD-box helicase RhlB is a component of the multienzyme RNA degradosome assembly, and its interaction with the core ribonuclease RNase E boosts the ATP-dependent activity of the helicase. Earlier studies have identified the regulator of ribonuclease activity A (RraA) as a potential interaction partner of both RNase E and RhlB. We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB. Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å. The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases.     

Role of SUV3 helicase in maintaining mitochondrial homeostasis in human cells

In yeast mitochondria, RNA degradation takes place through the coordinated activities of ySuv3 helicase and yDss1 exoribonuclease (mtEXO), whereas in bacteria, RNA is degraded via RNaseE, RhlB, PNPase, and enolase. Yeast lacking the Suv3 component of the mtEXO form petits and undergo a toxic accumulation of omega intron RNAs. Mammalian mitochondria resemble their prokaryotic origins by harboring a polyadenylation-dependent RNA degradation mechanism, but whether SUV3 participates in regulating RNA turnover in mammalian mitochondria is unclear. We found that lack of hSUV3 in mammalian cells subsequently yielded an accumulation of shortened polyadenylated mtRNA species and impaired mitochondrial protein synthesis. This suggests that SUV3 may serve in part as a component of an RNA degradosome, resembling its yeast ancestor. Reduction in the expression levels of oxidative phosphorylation components correlated with an increase in reactive oxygen species generation, whereas membrane potential and ATP production were decreased. These cumulative defects led to pleiotropic effects in mitochondria such as decreased mtDNA copy number and a shift in mitochondrial morphology from tubular to granular, which eventually manifests in cellular senescence or cell death. Thus, our results suggest that SUV3 is essential for maintaining proper mitochondrial function, likely through a conserved role in mitochondrial RNA regulation.