Summary of currently identified RNA thermometers
Abstract
RNA thermometers (RNATs) are cis-encoded regulatory elements that modulate translational efficiently in response to environmental temperature. Since their initial discovery, numerous RNATs have been identified and characterized, with the majority of currently known RNATs present in a wide variety of bacterial species. RNATs repress translation at relatively low temperatures by physically preventing binding of the ribosome to the regulated transcript by incorporating the Shine-Dalgarno sequences (and/or start codon) into an inhibitory structure. As the environmental temperature increases, the inhibitory structure within the RNAT is destabilized and the repression of translation initiation is gradually relieved. With the development of identification techniques, the rate at which RNATs are identified, and the understanding of the molecular mechanisms governing their regulator function, has grown exponentially. With the ever-increasing number of characterized RNATs, broad families of these regulators have now been identified. It has also become abundantly clear that RNATs influence several essential physiological processes. This chapter aims to summarize the current knowledge of bacterial RNATs, with special emphasis placed on the molecular mechanisms underlying RNAT function, experimental techniques used to identify and characterize RNATs, families of bacterial RNATs, as well as biological processes controlled by RNATs, and future directions of the field.
Keywords
- RNA thermometer
- ribo-regulator
- gene regulation
- heat shock response
- virulence factors
1. Introduction
Whether it is within a host or within the non-host environment, bacteria experience frequent, and often extreme, changes within their immediate environment. In order to survive and thrive under different environmental conditions, bacteria have evolved various systems that function to sense changes in environmental conditions and mediate rapid adaptation in response to the specific change. One condition that varies between the different environments encountered by pathogenic and non-pathogenic bacteria alike is temperature. Environmental temperature has direct effects on several fundamental biological processes, including proper folding of proteins and optimum activity of enzymes. To counteract the potentially detrimental effects of altered temperature, bacteria have evolved several strategies to respond to changes in environmental temperature, including specific heat shock and cold shock responses. Moreover, for pathogenic bacteria, a change of environmental temperature is a critical cue that can indicate entry into the host and/or progression of the disease process within an infected host. In order to establish and progress an infection, bacteria not only need to efficiently adapt to changing environmental conditions but also need to precisely regulate the production of specific virulence factors — processes that are dependent on the ability of bacteria to sense specific changes in environmental conditions, including temperature.
One method of sensing alterations in environmental temperature is through changes in the secondary structure of RNA molecules. Double-stranded regions within a given RNA molecule tend to dissociate into single-stranded structures with an increase in environmental temperature. The temperature at which half of the population of a given double-stranded RNA molecule is in the single-stranded conformation is defined as the Tm, a feature that is commonly used as a measurement for the stability of a given structure within an RNA molecule [1]. Due to its propensity to change conformation, an RNA structure that has a relatively low Tm is more responsive to changes in environmental temperature, a feature that facilitates its potential to act as a molecular thermosensors [2].
It is well established that translational efficiency is affected by the secondary structure of an RNA transcript, particularly that of the region containing the ribosome-binding site and/or start codon [3]. It was not until 1989, however, that the first
With the ever-increasing number of characterized RNATs, variability within this class of regulators is now coming to light. While the majority of RNATs are composed of sequences within the 5′ untranslated region (5′ UTR) of the regulated gene, some have now been shown to be composed, at least in part, of sequences within the coding region of the regulated transcript or by sequences within the coding region of a preceding gene within a polycistronic transcript [8,9]. In addition, the number of stem loops composing different RNATs varies, ranging from one in the simplest RNATs to five in the most complex RNATs [10,11]. Despite the variability among RNATs, they all share several basic fundamental features. Identifying and understanding the functional contribution of features conserved among characterized RNATs, as well as those that vary among this class of regulators, has and will continue to inform the foundational knowledge of the biological functions and chemical nature of these ubiquitous regulators. This chapter focuses on bacterial RNATs and provides a comprehensive summary of the current state of knowledge of RNATs, with emphasis given to discussions of the molecular mechanism underlying RNAT function, experimental techniques used to identify and characterize RNATs, families of bacterial RNATs, as well as the biological processes controlled by RNATs, and future directions of the field.
2. Molecular mechanism underlying the regulatory function of RNA thermometers
The molecular mechanism underlying the regulatory activity of RNATs is exquisitely simple, mediated entirely by temperature-induced structural changes within a target mRNA molecule. The currently proposed model of the molecular mechanism underlying RNAT function is that of a zipper [5]. More specifically, at relatively low “non-permissive” temperatures, an inhibitory structure is formed within the RNAT, at least in part, by binding of Shine-Dalgarno (SD) sequences with upstream sequences within the regulated transcript. Once formed, the inhibitory structure functions to block translation initiation by physically preventing binding of the ribosome to the regulated transcript. With an increase of temperature to that within a permissive range, the base-pairs that stabilize the inhibitory structure within the RNAT gradually dissociate, the ribosome-binding site becomes increasingly exposed and translation proceeds (Figure 1) [7,12].
Though responsive to temperature, RNAT-mediated regulation is not an all-or-nothing regulation but rather the shifting of an equilibrium towards an open or closed configuration depending on temperature [5]. Furthermore, mutagenesis-based experimentation has clearly demonstrated that it is the altered stability of the inhibitory structure rather than the primary sequence that plays the most critical role in the regulatory function of RNATs [13].
Several features differentiate RNATs from metabolites-binding riboswitches, a superficially related class of ribo-regulators. Firstly, as demonstrated by UV and NMR spectroscopy assays, the temperature-induced destabilization of the inhibitory structure within a given RNAT is a gradual and reversible process [7,14]. As a result of these fundamental features, RNATs mediate a graded response to temperature as opposed to an “on/off” type of regulation that is often associated with riboswitch-mediated regulation. Secondly, unlike riboswitches, structural changes within the RNAT are not mediated by an interaction with a small molecule or other cellular component, a foundational feature confirmed by
While many advances have been made in recent years, several questions remain regarding the details of the molecular mechanism(s) underlying the activity of RNATs. For example, several studies investigating the regulatory mechanism of RNATs focus exclusively on the hairpin containing the SD sequence. As a consequence, the impact of additional structural features within an RNAT, particularly that of commonly observed upstream hairpins, remains largely unknown. Additionally, a recent study revealed that, for at least a subset of RNATs, the ribosome can bind to the SD sequence of the regulated transcript even at non-permissive temperatures when the inhibitory structure would be present [16]. Finally, while the current model of regulation invokes nothing more than temperature in mediating the structural changes that underlie the regulatory activity of RNATs, the role of additional factors, including that of the ribosome itself, remains the subject of active investigation.
3. Identification of RNA thermometers
3.1. In silico predictions
Given that the function of an RNAT is dependent on its structure, the identification of a new RNAT often starts with
3.2. Identification with experimental approaches
Regardless of the approach used to predict the existence of a functional RNAT, the thermosensing regulatory activity of each putative element must be validated experimentally. There are several lab-based approaches currently being utilized to demonstrate the functionality of newly identified RNATs. One way in which the thermoresponsive regulatory activity of a putative RNAT is tested is to clone the element being investigated between a constitutive or an arabinose-inducible plasmid promoter and a reporter gene (e.g.,
To further validate the functionality of a predicted RNAT,
Moreover, techniques that study the physical properties of an RNA molecule, such as the nuclear magnetic resonance (NMR) spectroscopy and UV melting analysis, can be utilized to investigate the detailed base-pairing and their changes in response to temperature, thus revealing structural information as well as the molecular basis of thermosensing [7,26]. Together, these experimental analyses provide information about the dynamics of the inhibitory structure of a putative RNAT.
In addition to studies aimed at characterizing temperature-dependent changes in secondary structure, the regulatory activity of putative RNATs can be verified using a toe-printing assay, an
3.3. RNA structuromics
Recently, a combination of experimental and next-generation high-throughput techniques have been used to identify the structures of every RNA molecule within a single organism, collectively termed the “RNA structurome” [28]. Structuromic analyses performed at various temperatures have the potential to reveal a massive amount of information that will directly lead to the discovery of potentially expansive numbers of temperature-responsive regulatory RNA elements including RNATs [2]. The structurome of
4. Families of RNA thermometers
The thermosensing activity of an RNAT is largely dependent on the physical features of its secondary structure, specifically by those features that impact the stability, or the Tm, of the inhibitory hairpin. In addition to the base-stacking interactions and the hydration shell of an RNA helix, other critical features of RNATs include 1) the number and stability of hairpins that are formed within the element; 2) the presence of canonical and non-canonical base-pairing within the inhibitory structure; 3) the existence of internal loops, bulges, or mismatches within the formed structure(s); and 4) the extent of base-pairing between sequences composing the SD site and/or start codon with upstream sequences contained on the transcript. Each of these features can directly impact the stability of the inhibitory structure within a given RNAT, which in turn dictates the responsiveness of the element to temperature. Despite sharing a common basic regulatory mechanism, differences in RNATs display different secondary structures and other key features, differences that are now used to classify bacterial RNATs into families. The two currently recognized families of RNATs are ROSE-like RNATs (repression of heat shock gene expression) and FourU RNATs. RNATs composing each of these two main families, as well as a few unique RNATs, are discussed below.
4.1. ROSE-like RNA thermometers
ROSE-like elements were first identified as conserved
The ROSE-like family is the most extensively studied family of RNATs, harboring approximately 70% of all RNATs identified to date. All RNATs within the ROSE-like family are housed with 5′ UTR regions that range from 60 nucleotides to more than 100 nucleotides in length and that form 2 to 4 hairpins [36,37]. Within these hairpins, the 5′-proximal hairpin generally acts to stabilize the secondary structure and facilitate the correct folding of the other hairpins, while the 3′-proximal hairpin contains the SD region of the regulated transcript [7]. The defining features of ROSE-like RNATs that contribute to their temperature-responsive regulatory function include 1) the presence of a conserved anti-SD sequence 5′-UYGCU-3′ (Y stands for a pyrimidine) in the 3′-proximal hairpin, and 2) a “bulged” guanine within the SD sequestering hairpin (Figure 5) [36]. As a feature shared by all ROSE-like elements, it has been proposed that the “bulged” guanine within the SD sequestering hairpin is essential for the thermoresponsiveness of the regulatory element, a prediction that is supported by various mutagenesis-based experimental approaches and by NMR spectroscopy [7,38,39]. These studies have not only demonstrated that the “budged” guanine is essential for function but also revealed that the “bulged” guanine forms hydrogen bonds with the second guanine within the SD sequence of 5′-AGGA-3′. Additionally, towards the 3′ end of the SD site, two pyrimidines from the anti-SD strand form a triple-base pair with a uracil from the SD site with hydrogen bonds (Figure 5). The existence of two highly unstable pairs — a G-G pair and a triple-base pair — within the inhibitory hairpin of ROSE-like RNATs enables it to respond to the subtle changes of environmental temperature and thus to function as a temperature-sensitive regulatory element [7].
4.2. FourU RNA thermometers
FourU RNATs, so named due to the presence of four consecutive uracil residues within the SD sequestering inhibitory hairpin, represent the second family of currently identified RNATs. First identified in
The structural features of FourU RNATs are largely varied. For example, the length of the 5′ UTRs in which FourU RNATs are housed ranges from as short as 40 nucleotides (
4.3. Additional types of RNA thermometers
It is important to note that not all characterized RNATs fit neatly into one of the two main families: ROSE-like and FourU. While all RNATs are thought to share a basic zipper-like thermosensing mechanism, several identified RNATs differ from those composing the main families in critical features, including primary sequence and/or secondary structure, features that impact the regulatory activity of these elements. It is the identification and characterization of the details of the molecular mechanisms underlying each of these additional types of RNATs that will expand our understanding of foundational principles governing RNA-mediated thermosensing.
In some RNATs, base-pairing involving the SD sequence is not complete but instead is disrupted by mismatches or “bulged” nucleotides, a feature also noted for ROSE-like elements. For example, the inhibitory structure within the RNATs that control the production of two putative lipoproteins LigA and LigB in
Although lacking the presence of four consecutive uracil residues, two RNATs are similar to FourU RNATs in that they display more than 5 continuous base-pairs within the SD region of their inhibitory hairpins: one RNAT controls the production of an sHsp (Hsp17) from
For some RNATs, the function and stability of the inhibitory hairpin are impacted by base-pairing with sequences other than those within the SD region. For example, in the 5′ UTR of
A unique example among currently identified RNATs is the one that controls the expression of
Lastly, there are currently three characterized RNATs that are located within intergenic regions of a polycistronic transcripts:
Although they display key features that differ from those possessed by RNATs in the ROSE-like or FourU families, many of the unique RNATs highlighted above are conserved between several bacterial species. There is little doubt that as additional bacterial RNATs are identified and characterized, commonalities will emerge and additional families will be recognized.
5. Bacterial processes controlled by RNA thermometers
The regulation of gene expression in response to changes in environmental temperature is important for survival of all bacteria and for virulence of pathogenic bacteria. RNATs have been found to confer efficient temperature-dependent regulation onto the expression of bacterial genes encoding factors involved in two critically important bacterial processes —heat shock response and virulence. In the following section, each of these two critical biological processes will be briefly introduced and the role that RNATs play in facilitating them will be discussed.
5.1. Heat shock response
The primary effect of increased temperature on bacteria is the resulting denaturation of temperature-sensitive proteins. Similar to regulatory RNA molecules, the function of a protein is strictly dependent on its structure, a feature that can be impacted by environmental temperature. Increased environmental temperature can result in partial or complete denaturation of a protein, resulting in a stable but often non-functional molecule [50]. In addition to the denaturation of proteins, high temperature is also associated with disruption of the bacterial cell membrane as well as damage to DNA molecules [46,51]. As a result of these effects, increased environmental temperature can be lethal to bacterial life and thus represents a stress that must be overcome.
In order to facilitate responsive adaptation to a rise in environmental temperature, bacteria express several genes that encode for factors that function to protect the organism from the detrimental effects generated by heat, collectively termed the heat shock response [52]. The main components of the heat shock response include 1) alternative sigma factors that direct the transcription of other heat shock responding genes; 2) heat shock proteins (Hsps), such as protein chaperon machinery that facilitate the proper folding of other proteins; 3) small Hsps (sHsps) that have multiple functions including preventing the formation of protein aggregates and protecting the integrity of cellular membrane; and 4) enzymes that degrade denatured proteins, repair damaged DNA, and more
Understanding the molecular mechanisms underlying the temperature-dependent regulation of factors that facilitate the bacterial heat shock response is a major focus of ongoing investigations; the discovery of RNATs is rooted in these important studies. Since the identification of an RNAT that regulates the expression of a small heat shock protein (HspA from
5.2. Virulence-associated genes of pathogenic bacteria
Once within the body of the host, and throughout the course of a natural infection, pathogenic bacteria face several challenges, including but not limited to 1) the need to adhere to host cells, 2) the need to evade killing by the host immune system, and 3) the need to acquire essential nutrients. To overcome these challenges and progress of an infection, bacteria produce specific virulence factors. As the production of virulence factors is most beneficial to an invading bacterium when it is within the host, several levels of regulation are often employed to ensure that the production of these important factors occurs only when the bacteria is within an environment that resembles that encountered within the infected host. RNATs are involved in regulating the production of a variety of virulence factors in several species of pathogenic bacteria, ensuring that these factors are most efficiently produced at the relatively high temperatures encountered within the infected host (Figure 7).
The expression of many virulence-associated genes is controlled by protein-based regulation, specifically that carried out by transcriptional regulators. Interestingly, RNATs have been found to directly control the production of three transcriptional activators that, in turn, function to control the expression of virulence-associated genes:
RNATs have also been implicated in controlling the expression of virulence-associated genes that encode factors involved in adhesion and immune evasion. For example, three virulence-associated genes in
To date, two genes involved in the acquisition of essential nutrients have been shown to be regulated by RNATs:
For many pathogenic bacteria, the transmission from one host to the next involves exposure to different environments with different temperatures. The expression of many virulence-associated genes is influenced by environmental temperature, a signal that varies between the host and non-host environments. With an increasing number of virulence-associated genes that are now known to be regulated by the activity of RNATs, it is possible that temperature-dependent regulation mediated by RNATs will emerge as one of the basic regulatory strategies utilized by pathogenic bacteria. The full and potentially expansive role that RNATs play in controlling virulence of pathogenic bacteria is yet to be revealed.
6. Future directions
Although RNA-dependent regulation of gene expression has been a topic of active investigation for decades, investigations of RNATs are much more recent, with less than 100 RNATs having been identified to date (Table 1). Of note, RNATs vary in key structural features and influence different essential physiological processes.
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ROSE-element |
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Small heat shock protein | Balsiger |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Narberhaus |
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Small heat shock protein | Nocker |
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Small heat shock protein | Waldminghaus |
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CC2258 & CC3592 | Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Nocker |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Krajewski |
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Small heat shock protein | Nocker |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Small heat shock protein | Waldminghaus |
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Enzymes involved in the production of biosurfactant rhamnolipids | Grosso-Becerra |
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FourU element |
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Stress-responding periplasmic protease | Klinkert et. al. 2012 [10] |
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Small heat shock protein | Waldminghaus |
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Stress-responding periplasmic protease (transcribed from the 3rd promoter of the gene) | Klinkert et. al. 2012 [10] | ||
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Outer membrane heme-binding protein | Kouse |
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Outer membrane heme-binding protein | Kouse |
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Transcriptional activator of virulence factors (including cholera toxin) | Weber et. al. 2014 [40] | |
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Transcriptional activator of multiple virulence genes | Böhme |
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Transcriptional activator of multiple virulence genes | Böhme |
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Additional types |
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Heat shock alternative sigma factor σ32 | Morita |
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Putative small heat shock proteins (similar to hspA,B,C) | Krajewski et. al. 2014 [8] | |
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Component of protein chaperon machinery | Cimdins |
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Small heat shock protein | Kortmann |
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Putative lipoproteins promote adhesion -virulence related | Matsunaga |
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Transcription activator of virulence factors | Johansson |
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Capsule biosynthesis | Loh [24] |
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Factor H binding protein | |||
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Lipopolysaccharide modification | |||
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Quorum sensing –synthesis quorum sensing signal | Grosso-Becerra |
Despite their differences, all currently characterized RNATs are thought to share the same basic zipper-like temperature-responsive molecular mechanism, based on which both experimental and therapeutic applications can be derived. For example, artificial RNATs that have only a single hairpin to perform the temperature-dependent inhibition of translation have now been designed [55]. These artificial RNATs can be used as genetic tools to manipulate target gene expression. In the aspect of applying knowledge of RNATs in developing therapeutics, it is conceivable that compounds can be developed that would specifically stabilize the inhibitory structure within a given RNAT, thus decreasing expression of this target gene. Utilizing such an approach to inhibit the production of an essential gene product or virulence factor could prevent or limit infections by a variety of pathogenic bacteria.
Future applications of RNATs as genetic tools and/or drug targets are dependent on an increased understanding of these ubiquitous regulatory elements. With the maturation and development of experimental techniques, we could identify additional RNATs and study the molecular mechanisms underlying their regulatory activity in even greater detail. Moreover, due to the fundamental roles of RNA in the biological world, there is a great potential that RNATs also exist in archaea and eukaryotes. Further investigation and characterization of the conserved features and mechanisms of RNATs along with an understanding of the function of their regulatory targets could provide insight into the complex evolution of gene regulation. With the rate at which advances have been made in the field of RNA-mediated regulation, and specifically within the study of RNATs, there is no doubt that these and other important findings will be revealed sooner than later.
7. Nomenclature
Acknowledgments
The authors would like to acknowledge both the Ohio University, Ohio University Heritage College of Osteopathic Medicine, and the American Heart Association for funding their ongoing studies of bacterial RNA thermometers.
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