Katarzyna J. Bandyra

The Seed Region of a Small RNA Drives the Controlled Destruction of the Target mRNA by the Endoribonuclease RNase E

Summary Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5′-end of the cognate-pairing “seed.” Moreover, in the absence of the target the 5′-monophosphate makes the sRNA seed region vulnerable to an attack by RNase E against which Hfq confers no protection. These results suggest that the chemical signature and pairing status of the sRNA seed region may help to both ‘proofread’ recognition and activate mRNA cleavage, as part of a dynamic process involving cooperation of RNA, Hfq and RNase E.

The social fabric of the RNA degradosome.

Bacterial transcripts each have a characteristic half-life, suggesting that the processes of RNA degradation work in an active and selective manner. Moreover, the processes are well controlled, thereby ensuring that degradation is orderly and coordinated. Throughout much of the bacterial kingdom, RNA degradation processes originate through the actions of assemblies of key RNA enzymes, known as RNA degradosomes. Neither conserved in composition, nor unified by common evolutionary ancestry, RNA degradosomes nonetheless can be found in divergent bacterial lineages, implicating a common requirement for the co-localisation of RNA metabolic activities. We describe how the cooperation of components in the representative degradosome of Escherichia coli may enable controlled access to transcripts, so that they have defined and programmable lifetimes. We also discuss how this cooperation contributes to precursor processing and to the riboregulation of intricate post-transcriptional networks in the control of gene expression. The E. coli degradosome interacts with the cytoplasmic membrane, and we discuss how this interaction may spatially organise the assembly and contribute to subunit cooperation and substrate capture. This article is part of a Special Issue entitled: RNA Decay mechanisms.

Recognition of the small regulatory RNA RydC by the bacterial Hfq protein

Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a ‘seed’ region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at a 3.48 Å resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein–RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target. DOI: http://dx.doi.org/10.7554/eLife.05375.001

Licensing and due process in the turnover of bacterial RNA

RNA enables the material interpretation of genetic information through time and in space. The creation, destruction and activity of RNA must be well controlled and tightly synchronized with numerous cellular processes. We discuss here the pathways and mechanism of bacterial RNA turnover, and describe how RNA itself modulates these processes as part of decision-making networks. The central roles of RNA decay and other aspects of RNA metabolism in cellular control are also suggested by their vulnerability to sabotage by phages; nonetheless, RNA can be used in defense against phage infection, and these processes are described here. Salient aspects of RNA turnover are drawn together to suggest how it could affect complex effects such as phenotypic diversity in populations and responses that persist for multiple generations.

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. DOI: http://dx.doi.org/10.7554/eLife.16413.001

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

Abstract 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.

Viral interference of the bacterial RNA metabolism machinery

ABSTRACT In a recent publication, we reported a unique interaction between a protein encoded by the giant myovirus phiKZ and the Pseudomonas aeruginosa RNA degradosome. Crystallography, site-directed mutagenesis and interactomics approaches revealed this ‘degradosome interacting protein’ or Dip, to adopt an ‘open-claw’ dimeric structure that presents acidic patches on its outer surface which hijack 2 conserved RNA binding sites on the scaffold domain of the RNase E component of the RNA degradosome. This interaction prevents substrate RNAs from being bound and degraded by the RNA degradosome during the virus infection cycle. In this commentary, we provide a perspective into the biological role of Dip, its structural analysis and its mysterious evolutionary origin, and we suggest some therapeutic and biotechnological applications of this distinctive viral protein.

RNase E and the High-Fidelity Orchestration of RNA Metabolism

The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced. Early in its evolutionary history, RNase E acquired a natively unfolded appendage that recruits accessory proteins and RNA. These accessory factors facilitate the activity of RNase E and include helicases that remodel RNA and RNA-protein complexes, and polynucleotide phosphorylase, a relative of the archaeal and eukaryotic exosomes. RNase E also associates with enzymes from central metabolism, such as enolase and aconitase. RNase E-based complexes are diverse in composition, but generally bear mechanistic parallels with eukaryotic machinery involved in RNA-induced gene regulation and transcript quality control. That these similar processes arose independently underscores the universality of RNA-based regulation in life. Here we provide a synopsis and perspective of the contributions made by RNase E to sustain robust gene regulation with speed and accuracy.

Central dogma alchemy

Reflecting on the results of studies of RNA from the last 20 years, one might gain the impression that RNA could be a protein in clever disguise. For decades, RNA had primarily occupied a place in the central dogma of molecular biology as a transient messenger that passively conveys cellular genetic information in a directional flow that starts with DNA, proceeds through RNA, and finally culminates in protein synthesis. Aside from that fleeting role, RNA was also perceived to make more permanent but passive structural contributions as a scaffold for the ribosome or to present anticodons in tRNA. Although these functions for RNA are certainly not entirely inaccurate, the past two decades have revealed that the nucleic acid plays numerous, information-rich, and versatile roles rivaling those often assigned to proteins. The picture emerging from the key advances in the last two decades is that RNA is a chemist, modulator of gene expression both pre- and post-transcription, component of computational networks, allosteric entity, signaling molecule, and an agent of acquired immunity. Regarding the features of RNA as a chemist, consider how chemical transformations are powerfully mediated by proteins in life-as-we-know it. But before life became so familiar, some of the key transformations that made life possible in the first place are likely to have been mediated by RNA. The work of the Steitz and Moore groups demonstrated in 2000 that the peptidyl transferase site consists of only ribosomal RNA, implicating the RNA in catalyzing peptide bond formation. More recent crystallographic work from the Steitz laboratory highlights the intricate chemistry of the process, involving channeling of protons in a “wire” supported by entrained water and mostly nucleic acid substituents. These observations put overwhelming stereochemical proof of a hypothesis advanced by Harry Noller and others, based on observations that protein-free ribosome preparations can still catalyze peptidyl transfer, that the ribosome is a ribozyme. Other natural ribozyme activities, such as capacity for phospho-transfer, have been illuminated in the last two decades with crystal structures of self-cleaving RNA from the laboratories of Scott, Pyle, and Doudna and many other groups. Piccirilli, Staley and colleagues have obtained evidence that the RNA in the spliceosome likely evolved from an RNA catalyst from the group II self-splicing intron. These advances, together with the discovery and structural characterization of artificially selected RNAs that can act as template-based polymerases, have delivered further support that RNA might have been a precursor of the life-as-we-know it. Admittedly, the chemical transformation of peptidyl and phospho-transfer mediated by RNA do not have the high catalytic powers of typical protein-based enzymes in life-as-we-know it; but still to be explored are the capacity of RNA catalysts with metal cofactors in reactions under the reducing conditions of early life, or in photochemical processes. Another key protein-like feature of RNA is its capacity to communicate allosteric signals and, perhaps somewhat overlooked, its potential for cooperative behavior. Cooperativity underlies the capacity of modern cells to respond rapidly to changes in environment, or to signal for genetic programs in metazoan embryogenesis, or social behavior in prokaryotes. An important allosteric effect of RNA is the ability to switch conformation upon binding effector molecules or in response to environmental changes, for instance acting as a thermometer with temperature-dependent structural states. The discovery and characterization of metabolite-responsive RNA “riboswitches” that are often a part of an mRNA, by the Breaker group and other laboratories, demonstrates RNA not being a passive carrier but rather an active entity that can change conformation induced by ligand binding, with functional consequences on rates of translation and degradation. Allosteric and cooperative effects play roles in the recruitment of binding partners in ribonucleoprotein complexes such as the splicing machinery, as illuminated by the structural work of Kiyoshi Nagai and others. RNA can also be an allosteric ligand for other RNAs, which in turn can communicate recognition signals for proteins. Given the capacity of RNA to be remodeled with functional consequence through interactions with metabolites, proteins, and other RNA, it is perhaps little wonder that the cells are replete with RNA helicases, which are energy-dependent machines that keep these interactions in a state of flux. Structural and functional studies of helicases over the last two decades suggest that dynamic remodeling might underlie sensitive responses to environmental conditions mediated by RNA allostery. The major discoveries in the RNA world over the past 20 years have been dominated by the structure of the ribosome and mechanism of protein translation. These have tremendously expanded the recognized repertoire of RNA secondary and tertiary folds. However, the RNA in the ribosome has turned out to be more than just the skeleton of this ribonucleoprotein; it is now seen to have allosteric behavior that is fundamental for communicating structural signals in almost every key step of protein synthesis. The structural data have illuminated how the cognate pairing of tRNA and mRNA at the codon recognition center triggers a chain of events that faithfully bring the correct amino acid at the peptidyl transfer center in the ribosome through allosteric activation of GTP hydrolysis. Structural deformations in the tRNA and ribosome, visualized by crystallography and cryoEM, are key for maintaining fidelity of translation. The structural and functional work on the ribosome has been a remarkable achievement with international efforts led by the laboratories of Thomas Steitz, Peter Moore, Venki Ramakrishnan, Ada Yonath, Harry Noller, and Marat Yusupov. New discoveries continue to be made through the rapid advances in cryoEM techniques that illuminate the remarkably conserved mechanism of peptide secretion through translocons. Another remarkable parallel between RNA and proteins, made clear over the last 20 years, is the capacity to participate in elaborate regulatory networks. Transcription factors are proteins that, through DNA binding or covalent modification of chromatin, make intricate and beautifully interconnected patterns that give complex control of gene expression. It is now becoming clear that RNAs can provide the same networking too, post-transcription. With the discovery of microRNAs in the 1990s, a re-examination commenced of short RNA fragments that were previously considered to be just cellular noise. Numerous classes of non-coding RNAs were identified that have key roles in regulation of gene expression in eukaryotes. Moreover, RNA mediated “riboregulation” was also found in bacteria, where it often helps in rapid adaptation to changes in the environment. Another set of key discoveries concerns the contribution of riboregulation to pre-transcriptional control, through activation of chromatin modification in promoter regions in eukaryotic genomes, or by regulating polymerase in bacterial (by physical sequestration of 6S RNA). RNA is also capable of modulating protein activities by physical sequestration, for instance in acting as an anti-toxin to a protein toxin. Many achievements in RNA biology were made possible by the development of RNA sequencing techniques. This led not only to better understanding of some biological processes in the investigated cell, but also to comparison of transcriptomes between, e.g., tissues or cellular lines grown under different conditions. With its widespread use currently, RNA sequencing has contributed to identification of gene expression patterns in response to environmental adaptation or in diseases. It has also proven to be a powerful tool for discovery through applications such as ribosome profiling, which has illuminated numerous features of translation including recognition of regulatory elements and control. Immunity was long thought to be the exclusive domain of proteins, which can be used to recognize diverse epitopes. In metazoans, viral RNA can be recognized by chemical signatures on the 5′ end and structure, such as recognition by the Rig-I helicase. RNA can thus be a signaling molecule to initiate innate immune response. A surprising discovery was that RNA can recognize foreign nucleic acids and confer acquired immunity against viruses in bacteria. The discovery of this system—the CRISPR system—was soon followed by applications for genome engineering, which is widely exploited in research, and could possibly be applied in medicine. We are moving from the perception that the information contained in RNA is mainly encoded in its linear sequence, but the emerging image of this molecule, accounting for the numerous roles it can play in the cell, shows that the sequence is only one element of complex entirety. RNA is no longer considered just a string of nucleotides, but rather as a structured molecule capable of catalyzing enzymatic reactions, interacting with and modulating its RNA and protein partners, as well as regulating many physiological processes. It is not only a template for protein synthesis, but protein-like formation, that forms a parallel to proteome network in the cell to help carry out and maintain the life-as-we-know it.

Stem-loops direct precise processing of 3′ UTR-derived small RNA MicL

Increasing numbers of 3′UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To understand the sequence and structure determinants for the precise processing of the 3′ UTR-derived sRNAs, we examined the cleavage of multiple mutant and chimeric derivatives of the 3′ UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3′ to the cleavage site define optimal, correctly-positioned cleavage of MicL and likely other similar sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.