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.     

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.     

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.     

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.     

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.     

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.     

Crystal Structure of Escherichia coli Polynucleotide Phosphorylase Core Bound to RNase E, RNA and Manganese: Implications for Catalytic Mechanism and RNA Degradosome Assembly

Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel β-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.     

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.     

The Crystal Structure of Hexamer RraA from Pseudomonas Aeruginosa Reveals Six Conserved Protein–Protein Interaction Sites

RNase E functions as the rate-limiting enzyme in the global mRNA metabolism as well as in the maturation of functional RNAs. The endoribonuclease, binding to the PNPase trimer, the RhlB monomer, and the enolase dimer, assembles into an RNA degradosome necessary for effective RNA metabolism. The RNase E processing is found to be negatively regulated by the protein modulator RraA which appears to work by interacting with the non-catalytic region of the endoribonuclease and significantly reduce the interaction between RNase E and PNPase, RhlB and enolase of the RNA degradosome. Here we report the crystal structure of RraA from P. aeruginosa to a resolution of 2.0 ?. The overall architecture of RraA is very similar to other known RraAs, which are highly structurally conserved. Gel filtration and dynamic light scattering experiments suggest that the protein regulator is arranged as a hexamer, consistent with the crystal packing of "a dimer of trimer" arrangement. Structure and sequence conservation analysis suggests that the hexamer RraA contains six putative charged protein-protein interaction sites which may serve as binding sites for RNase E.     

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.     

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.     

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 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.     

The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP-stabilizers

In Escherichia coli, REP-stabilizers are structural elements in polycistronic messages that protect 5'-proximal cistrons from 3'-->5' exonucleolytic degradation. The stabilization of a protected cistron can be an important determinant in the level of gene expression. Our results suggest that RNase E, an endoribonuclease, initiates the degradation of REP-stabilized mRNA. However, subsequent degradation of mRNA fragments containing a REP-stabilizer poses a special challenge to the mRNA degradation machinery. Two enzymes, the DEAD-box RNA helicase, RhlB and poly(A) polymerase (PAP) are required to facilitate the degradation of REP-stabilizers by polynucleotide phosphorylase (PNPase). This is the first in vivo evidence that these enzymes are required for the degradation of REP-stabilizers. Furthermore, our results show that REP degradation by RhlB and PNPase requires their association with RNase E as components of the RNA degradosome, thus providing the first in vivo evidence that this ribonucleolytic multienzyme complex is involved in the degradation of structured mRNA fragments.     

Nucleic acid and protein factors involved in Escherichia coli polynucleotide phosphorylase function on RNA

It has been reported that polynucleotide phosphorylase (PNPase) binds to RNA via KH and S1 domains, and at least two main complexes (I and II) have been observed in RNA-binding assays. Here we describe PNPase binding to RNA, the factors involved in this activity and the nature of the interactions observed in vitro. Our results show that RNA length and composition affect PNPase binding, and that PNPase interacts primarily with the 3′ end of RNA, forming the complex I-RNA, which contains trimeric units of PNPase. When the 5′ end of RNA is blocked by a hybridizing oligonucleotide, the formation of complex II-RNA is inhibited. In addition, PNPase was found to form high molecular weight (>440 kDa) aggregates in vitro in the absence of RNA, which may correspond to the hexameric form of the enzyme. We confirmed that PNPase in vitro RNA binding, degradation and polyadenylation activities depend on the integrity of KH and S1 domains. These results can explain the defective in vivo autoregulation of PNPase71, a KH point substitution mutant. As previously reported, optimal growth of a cold-sensitive strain at 18 °C requires a fully active PNPase, however, we show that overexpression of a novel PNPaseΔS1 partially compensated the growth impairment of this strain, while PNPase71 showed a minor compensation effect. Finally, we propose a mechanism of PNPase interactions and discuss their implications in PNPase function.     

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.