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 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.     

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.     

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.     

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 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.     

Hfq binding at RhlB-recognition region of RNase E is crucial for the rapid degradation of target mRNAs mediated by sRNAs in Escherichia coli

An RNA chaperon Hfq along with Hfq-binding sRNAs stably binds to RNase E in Escherichia coli. The role of the Hfq-RNase E interaction is to recruit RNase E to target mRNAs of sRNAs resulting in the rapid degradation of the mRNA-sRNA hybrid. The C-terminal scaffold region of RNase E is responsible for the interaction with Hfq. Here, we demonstrate that the scaffold region can be deleted up to residue 750 without losing the ability to cause the rapid degradation of target mRNAs mediated by Hfq/sRNAs. The truncated RNase E750 can still bind to Hfq although the truncation significantly reduces the Hfq-binding ability. We conclude that the subregion between 711 and 750 is sufficient for the functional interaction with Hfq to support the rapid degradation of ptsG mRNA although additional subregions within the scaffold are also involved in Hfq binding. Deletion of the 702-750 region greatly impairs the ability of RNase E to cause the degradation of ptsG mRNA. In addition, a polypeptide corresponding to the scaffold region binds to Hfq without the help of RNA. Finally, we demonstrate that overexpression of RhlB partially inhibits the Hfq binding to RNase E and the rapid degradation 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.     

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.     

Chloroplast PNPase exists as a homo-multimer enzyme complex that is distinct from the Escherichia coli degradosome.

In Escherichia coli, the exoribonuclease polynucleotide phosphorylase (PNPase), the endoribonuclease RNase E, a DEAD-RNA helicase and the glycolytic enzyme enolase are associated with a high molecular weight complex, the degradosome. This complex has an important role in processing and degradation of RNA. Chloroplasts contain an exoribonuclease homologous to E. coli PNPase. Size exclusion chromatography revealed that chloroplast PNPase elutes as a 580-600 kDa complex, suggesting that it can form an enzyme complex similar to the E. coli degradosome. Biochemical and mass-spectrometric analysis showed, however, that PNPase is the only protein associated with the 580-600 kDa complex. Similarly, a purified recombinant chloroplast PNPase also eluted as a 580-600 kDa complex after gel filtration chromatography. These results suggest that chloroplast PNPase exists as a homo-multimer complex. No other chloroplast proteins were found to associate with chloroplast PNPase during affinity chromatography. Database analysis of proteins homologous to E. coli RNase E revealed that chloroplast and cyanobacterial proteins lack the C-terminal domain of the E. coli protein that is involved in assembly of the degradosome. Together, our results suggest that PNPase does not form a degradosome-like complex in the chloroplast. Thus, RNA processing and degradation in this organelle differ in several respects from those in E. coli.     

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.     

RNaseE and RNA helicase B play central roles in the cytoskeletal organization of the RNA degradosome

The RNA degradosome of Escherichia coli is a multiprotein complex that plays an essential role in normal RNA processing and decay. It was recently shown that the major degradosome constituents are organized in a coiled cytoskeletal-like structure that extends along the length of the cell. Here we show that the endoribonuclease E (RNaseE) and RNA helicase B (RhlB) components of the degradosome can each independently form coiled structures in the absence of the other degradosome proteins. In contrast, the cytoskeletal organization of the other degradosome proteins required the presence of the RNaseE or RhlB coiled elements. Although the RNaseE and RhlB structures were equally competent to support the helical organization of polynucleotide phosphorylase, the cytoskeletal-like organization of enolase occurred only in the presence of the RNaseE coiled structure. The results indicate that the RNA degradosome proteins are components of the bacterial cytoskeleton rather than existing as randomly distributed multiprotein complexes within the cell and suggest a model for the cellular organization of the components within the helical degradosomal structure.