RNA remodeling and gene regulation by cold shock proteins

Abstract

One of the many important consequences that temperature down-shift has on cells is stabilization of secondary structures of RNAs. This stabilization has wide-spread effects, such as inhibition of expression of several genes due to termination of their transcription and inefficient RNA degradation that adversely affect cell growth at low temperature. Several cold shock proteins are produced to counteract these effects and thus allow cold acclimatization of the cell. The main RNA modulating cold shock proteins of E. coli can be broadly divided into two categories, (1) the CspA family proteins, which mainly affect the transcription and possibly translation at low temperature through their RNA chaperoning function and (2) RNA helicases and exoribonucleases that stimulate RNA degradation at low temperature through their RNA unwinding activity.

Introduction: The Cold Shock Response

Living organisms encounter various stresses, change in temperature being one of the most common. Fluctuations in temperature have widespread effects on the growth and survival of bacteria, which have therefore developed mechanisms that allow them to adapt to these changes. The cold-shock response is one such mechanism; it consists of a number of adaptive changes ranging from alterations in membrane composition to alterations in the global protein profile of the cell. Physiologically, the cold shock response of a bacterium like Escherichia coli manifests itself as an acclimation phase during which cell growth completely (but temporarily) stops immediately after the temperature downshift. During this phase, several cold shock proteins, which help the cells to adopt to low temperature, are produced. After the acclimation phase is over, the synthesis of most of the cold shock proteins decreases to a new basal level and synthesis of non-cold shock proteins resumes, allowing cells to grow at low temperature, albeit at a slower rate (review in ref. 1–3). Certain bacteria such as B. subtilis and Lactobacillus lactis do not exhibit a temporary cessation of growth upon cold-shock response but instead immediately proceed to slow-rate low-temperature growth.4,5 These bacteria might be “preconditioned” to low temperature growth even when growing at their physiologically optimal temperature. Cold shock proteins play a critical role in the cold shock response of E. coli. Some of the main cold shock proteins of E. coli are the RNA chaperones CspA,6 CspB,7 CspG8 and CspI,9 RNA helicase CsdA,10 ribosome-binding factor RbfA,11 polynucleotide phosphorylase (PNPase),12–14 transcription factor NusA,15 translation initiation factors IF1,16 and IF2,17 RecA,18 histone-like protein H-NS,19 DNA gyrase,20 pY,21 protein chaperones, Trigger factor,22 Hsc-66,23 HscB,23 ClpB,24 Recombination factor, dihydrolipoamide transferase,25 pyruvate dehydrogenase,25 and trehalose synthesizing proteins.22 The relative amounts of all these proteins (and, doubtless, many others) are increased upon temperature down shift. However, time-resolved analysis of cells undergoing low temperature acclimation reveals a clear sequence of appearance of various cold shock proteins, indicating that the cold shock response is hierarchical, and some proteins are needed at the earliest stages of cold shock response, while others are needed at later stages and may be regulated by proteins of the first kind.

Sensing the Temperature Change at Cellular Level

The cell uses various thermosensors, including membranes, nucleic acids, proteins etc., to register temperature changes (reviewed in ref. 26). Some of the major direct consequences of temperature downshift include (i) decrease in membrane fluidity affecting membrane-associated cellular functions such as active transport and protein secretion, (ii) changes in nucleic acid structures, (iii) reduced ribosome function which affects translation, especially of non-cold-shock proteins and (iv) inefficient protein folding. Various mechanisms are adapted by the cell to restore the flexibility of membranes for example, increasing proportion of unsaturated fatty acids (UFAs). The cis-vaccenic acid, with low melting point and high flexibility (homeoviscous adaptation) is one such fatty acid,27 which is formed from palmitoleic acid mediated by the enzyme β-ketoacyl-acyl carrier protein (ACP) synthase II. This enzyme is activated by cold shock.28,29 In the case of B. subtilis and cyanobacteria, cold-inducible desaturases play an important role in alteration of the degree of saturation of fatty acids in membrane phospholipids.30,31 Cold-induced changes in the structure of nucleic acid are of two types, (a) stabilization of the secondary structures of RNA and DNA and (b) increased negative supercoiling of DNA. The increase in negative supercoiling of DNA that occurs upon cold shock affects DNA related functions, such as replication, transcription and recombination.32–34 Stabilization of the secondary structures in RNAs, affects transcription and translation due to hindered movement of RNA polymerase and ribosomes, respectively. RNAs have been proposed to act as cellular thermometers.26,35,36 Certain E. coli proteins such as CspA, the main cold shock protein, and its homologs act as RNA chaperones37,38 melting the secondary structures in nucleic acids and facilitating transcription and translation at low temperature. This activity of CspA and its homologs is essential for cold acclimation of cells.39,40 Since CspA is among the first (and by far, most abundant) proteins induced upon cold shock, its RNA chaperoning function is considered as one of the main mechanism(s) of cold adaptation. Temperature downshift renders ribosomes nonfunctional in translation of most cellular mRNAs. However, this effect is not as severe for mRNAs of genes encoding cold-shock proteins. This selectivity is due to structural elements present in cold-shock proteins mRNAs that promote translation initiation at low temperature. During the growth lag period after the temperature downshift, cold-shock ribosomal factors such as RbfA and CsdA are produced. These proteins bind ribosomes, converting them to cold-adapted ribosomes that are capable of translating non-cold-shock mRNAs.41

Temperature downshift also leads to changes in certain proteins for example, aspartate chemoreceptor (Tar) of E. coli. These changes include reversible methylation of cytoplasmic signaling/adaptation domain.42 The temperature downshift also causes protein mis-folding, although it is not as severe as that caused by heat shock.43 DNA microrarray analysis of transcripts of E. coli cells subjected to cold shock showed that certain molecular chaperones such as Caseinolytic proteases (Clps), trigger factor and GroEL and GroES39 were induced upon cold shock. Trigger factor (TF) is induced at a modest level 2–3 h after cold shock.22 TF is a peptidyl prolyl isomerase that catalyzes the cis/trans isomerization of peptide bonds which are N-terminal to the proline residue.43 TF can associate with ribosomes and plays an important role in co-translational protein folding.44,45 Cell viability at 4°C showed direct correlation with the intracellular levels of TF, which is presumably due to its ability to help protein synthesis and folding at low temperature.43

RNA Modulating Cold Shock Proteins

Proteins that restructure RNAs are of different types (reviewed in ref. 46): (i) RNA chaperones that melt misfolded RNA molecules and thus promote proper folding, (ii) RNA annealers that accelerate annealing of complementary RNAs, (iii) RNA helicases that resolve the RNA structures using ATP hydrolysis and (iv) specific RNA binding proteins that can also contribute to RNA folding by stabilizing specific RNA structures. Here, we focus only on RNA modulating cold shock proteins of E. coli. The main RNA modulating cold shock proteins of E. coli can be broadly divided into two categories, (i) the CspA family proteins, which mainly affect DNA transcription and possibly RNA translation at low temperature (RNA chaperones), (ii) the helicases and exoribonucleases, which modulate the RNA metabolism at low temperature. The two types of proteins are schematically depicted in Figure 1.

The E. coli CspA Family

E. coli contains a family of nine homologous proteins (CspA to CspI), out of which only CspA, CspB, CspG and CspI are cold-shock inducible. A number of diverse and seemingly unrelated functions have been attributed to CspA and its homologs in different bacteria, such as, RNA chaperones,38 improved adaptation to cold-shock, stationary phase, nutrient starvation and freezing,47,48 antibiotic biosynthesis,49 UV sensitivity,50 regulation of expression of proteins responding to osmotic stress, oxidative stress and stationary phase (UspA, OsmY, Dps, ProP and KatG51), camphor resistance and chromosome condensation,52,53 downregulation of λ Q-mediated transcription antitermination,54 downregulation of poly(A)-mediated 3′ to 5′ exonucleolytic decay by PNPase,55 inhibition of DNA replication,56 and tolerance to solvents such as toluene.57

The cold-shock induction of CspA and its homologs was studied in detail and was shown to occur at the levels of transcription, mRNA stability and translation.

Regulation at the level of transcription.

The cspA transcript undergoes a 4–5-fold increase upon cold shock as revealed by studies using reporter gene fusions the reporter genes,58,59 primer extension, northern blot analysis and DNA microarray analysis of the global transcript profile of cold-shocked cells.39 No additional factors (other than cold) are necessary for cspA induction. The 15°C induction of cspA is observed even when the gene is fused to non-cognate promoter, however, the level of such heterologous induction is significantly less than that observed with the cognate cspA promoter. Certain elements seem to enhance the level of cspA promoter activity. These include (i) an AT-rich sequence (UP element) immediately upstream of the -35 region;58,59 and (ii) an extended -10 box a TGn motif preceding the -10 box. However, none of these elements seem to specifically contribute to low-temperature activity of the cspA promoter.

The cspA, cspB, cspG and cspI genes possess a long 5′ untranslated region (5′-UTR). This region contains a highly conserved, 11-base sequence termed the ‘cold box’. It has been reported that at high concentrations of CspA, the protein binds the cold-box and thus regulates its own expression. The overproduction of 5′UTR leads to prolonged synthesis of CspA, an effect suppressed by co-overproduction of CspA.60,61

Although CspA is the main cold shock protein, it is also overproduced, albeit at much lower levels, at 37°C during early exponential growth phase. This induction results from the location of the cspA gene near the oriC replication origin, which leads to higher gene dosage and higher stability of cspA mRNA due to lower RNase activity at this stage of growth.62 For similar reasons, 37°C production of CspA is observed upon nutritional upshift.63

Regulation at the level of stabilization of mRNA.

The cspA mRNA is transiently and dramatically stabilized immediately following cold shock. This stabilization is likely the major factor that leads to dramatic induction of CspA at low temperature.64 The 5′-UTR was shown to be responsible for the extreme instability of cspA mRNA at 37°C (a half-life of 12 s) and has a positive effect on its stabilization upon cold shock (a half-life of more than 20 min).59 The cspA promoter is active at 37°C, but the steady-state levels of cspA RNA and CspA synthesis are low at this temperature due to extreme instability of its mRNA. Using enzymatic and chemical probing, it was recently shown that the cspA mRNA undergoes a temperature-dependent structural rearrangement at low temperature, likely resulting from stabilization of an otherwise thermodynamically unstable folding intermediate. The “low temperature” structure is more efficiently translated and somewhat less susceptible to degradation than the 37°C structure.65 Thus, the cspA mRNA acts as a thermometer, sensing temperature downshifts by adopting a functionally distinct structure.65

Regulation at the level of translation.

The mRNAs of cold-inducible CspA homologs and those of ribosome-associated factors like csdA and rbfA, contain an A/T rich sequence located 14-bases downstream of their initiation codons. This element is called Translation-Enhancing Element and is presumed to enhance translation initiation in cold-shock mRNAs.66 As this sequence is complementary to a region in the penultimate stem of 16S rRNA, it was previously thought to help translation initiation by facilitating the formation of translation preinitiation complex through direct interaction with 16S rRNA. However, this view is not universally accepted and the exact mechanism of the stimulatory effect of TEE on translation initiation remains unknown at present.59,67

CspA homologs as transcription antiterminators.

The low-temperature-induced stabilization of the secondary structures of RNA interferes with both transcription and translation elongation. CspA homologs are proposed to act as RNA chaperones by facilitating transcription and translation at low temperature by virtue of their ability to ‘melt’ the secondary structures in nucleic acids. The function of CspA homologs as RNA chaperones promoting transcription antitermination has been studied in considerable detail.37,38,40 Modulation of transcription termination by RNA-binding proteins involves resolving hairpin structures in nascent RNA that can act as transcriptional terminators or pause sites, thus leading to transcript elongation (reviewed in ref. 68). The three-dimensional structures of CspA from E. coli and CspB from B. subtilis have been resolved by X-ray crystallography and NMR-analysis and found to be very similar.69–73 The protein consists of five antiparallel β-strands (β1 to β5) that form a β-barrel structure with two β-sheets. Two RNA-binding motifs, RNP1 and RNP2, are located on the β2 and β3 strands, respectively. The proteins have an overall negative surface charge with positively charged amino acids surrounding a surface-exposed aromatic patch (RNP1 W¹¹, F¹⁸ and F²⁰ and RNP2 F³¹, H³³ and F³⁴). After the initial approach to an RNA molecule through electrostatic attraction and subsequent binding through stacking of the aromatic RNP side chains with RNA bases, further intramolecular or intermolecular base pairing by a segment of RNA bound to a Csp protein is prevented by charge repulsion.74 CspA and its homologs do not exhibit high degree of specificity for their RNA/DNA substrates.38,75,76 The RNA chaperone activity allows the CspA homologs to act as transcription antiterminators and thus aid in cold acclimation of cells.37,40 CspE, a member of this family, was used as a model to analyze these activities. CspE caused transcription antitermination at every rho-independent terminator tested. Systematic mutations of individual amino acids of CspE showed that only Phe17, Phe30 and His32 are essential for nucleic acid melting activity. The Phe residues initiate the melting process and the His residue completes it. The proteins carrying substitutions of these amino acids with Arg fully retain their nucleic acid binding activity, but lose the ability to cause transcription antitermination and cold acclimation of cells as they lack the melting activity.40,77 DNA microarray analysis of the cold shock response of the wild-type cells, the cells carrying deletion of various csp genes along with the cells overexpressing the wild-type or melting-deficient Csp proteins revealed genes, which require the transcription antitermination activity of Csp proteins for expression at low temperature. The genes which showed strict dependence on Csp proteins for their expression at 15°C, included malE and malK (membrane related functions), mopA and mopB (chaperones), dps, katG, rpoS, uspA (stress response), as well as several others. Several of these genes possess promoter-proximal sequences that contain stem-loop structures, which could stabilize at low temperature and block transcript elongation. These sequences, termed Csp-responsive transcription terminators, are proposed to be physiological targets of Csp proteins. Csp-dependent melting of these structures is proposed to allow transcription of downstream genes to continue at low temperature.78

Helicases and Exoribonucleases

SrmB and CsdA, the DEAD-box helicases involved in cold shock acclimation.

The DEAD-box RNA helicase family proteins play important roles in many cellular processes such as processing, transport or degradation of RNA or ribosome biogenesis (reviewed in ref. 79). The E. coli encodes five DEAD-box RNA helicase family proteins, CsdA, DbpA, RhlB, RhlE and SrmB.80 SrmB and CsdA seem to play some role in the cold acclimation of cells and are briefly described below.

SrmB.

SrmB is involved in ribosome biogenesis. In fact, the srmB gene was originally isolated as a multicopy suppressor of mutations in the ribosomal protein L24 gene rplX.81 Deletion of srmB results in (i) slow-growth phenotype at low temperature, (ii) deficit in free 50S ribosomal subunits and (iii) accumulation of a new ribosomal particle sedimenting around 40S. Thus, it was suggested that there is a step of 50S assembly, which involves a structural rearrangement that, at least at low temperature, requires SrmB.82

CsdA.

The requirement of CsdA at low temperature is more pronounced than that of SrmB and the deletion of csdA gene severely impairs growth at low temperature.83,84 CsdA has been assigned multiple cellular functions including ribosome biogenesis, translation initiation and degradation of mRNAs.

The possibility of CsdA being involved in the biogenesis of ribosomal subunits was first suggested by an observation that its gene, deaD/csdA, can act as a multicopy suppressor of a missense mutation in the rpsB gene that encodes the ribosomal protein S2.10 Further, overexpression of CsdA restores the incorporation of mutant S2 (as well as the S1 protein) into 30S ribosomal subunits.85 Recently, it was shown that CsdA is also involved in the biogenesis of the 50S ribosomal subunits79 and associates with 50S precursors at low temperature.83

CsdA is homologous to eukaryotic translation initiation factor eIF4A. EIF4A catalyzes ATP-dependent unwinding of RNA duplexes and stimulates translational initiation. It was thus suggested that CsdA too may be involved in assisting the translation by promoting translation initiation of structured mRNAs.86

The role of CsdA in mRNA degradation was suggested by the observations that it (i) is found in degradosomes in cold-adapted cultures,87–89 and (ii) is involved in efficient and selective degradation of Csp mRNAs by unwinding the mRNA secondary structure that impedes the processive activity of PNPase.87 Although CsdA was shown to be involved in important physiological processes, it was not clear which of its partial activities (or their combination) play a role in its essential cold-shock function. Our data suggest that the main way through which CsdA contributes to cold acclimation is through its involvement in mRNA decay. The helicase activity of CsdA is pivotal for promoting the degradation of mRNAs stabilized at low temperature: helicase-deficient CsdA mutants do not complement cold sensitivity of the csdA deletion cells. The correlation between the helicase activity of CsdA and stability of mRNAs of cold-inducible genes was further shown using the cspA mRNA, which was significantly stabilized in the ΔcsdA cells. Again, this effect was counteracted by overexpression of the wild-type CsdA, but not helicase-deficient CsdA mutants.90 We used csdA deletion cells and screened an E. coli genomic library for protein(s) that can complement the essential CsdA function during cold acclimation. We observed that another DEAD-box RNA helicase, RhlE, complemented the csdA deletion.90 Other groups reported similar findings.79,91

CsdA and SrmB share several properties: they (i) unwind nucleic acid duplexes with 3′ or 5′ extensions, (ii) stabilize certain mRNAs,92 (iii) bind to RNase E88, (iv) participate in 50S assembly, probably by modulating RNA structures through their unwinding activity and (v) act as RNA chaperones that prevent and/or resolve misfolding. Despite these similarities, overexpression of SrmB does not suppress the cold sensitivity of the csdA deletion mutant and vice versa.83 The reasons for this specificity remain presently unknown.

Cold-inducible Exoribonucleases

RNase R.

PNPase, RNase R and RNase II are the three major 3′-to-5′ processing exoribonucleases in E. coli. These enzymes are primarily involved in RNA metabolism. Both PNPase,93,94 and RNase R95 are induced by cold shock and are suggested to be the universal degraders of structured RNA in the cell.96,97 RNase II is not cold shock inducible. Interestingly, RNase II, but not RNase R can complement the cold shock function of PNPase.98 RNase II and RNase R belong to the RNR family. Based on sequence analysis and comparison with the RNase II structure,99–101 RNase R consists of a central nuclease domain, two cold-shock (CSD) domains near the N-terminal region of the protein, an S1 domain and a highly basic C-terminal region.102

While not detected in our complementation screen of csdA deletion strain (above) overproduction of RNase R or CspA complemented the cold-sensitive phenotype of the deletion strain.90 The fact that CspA is an RNA chaperone and RNase R is an exoribonuclease further supported the hypothesis that the primary role of CsdA in cold acclimation is in mRNA decay and its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature.90 Interestingly, in Bacillus subtilis it was shown that cold-induced helicases and Csps work in together to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures.103

PNPase and RNase II overproduction did not complement CsdA helicase mutants.90 This observation led us to hypothesize that complementation of the cold-shock function of CsdA by RNase R is not merely due to its ability to degrade secondary structures in RNAs, but instead may be due to a helicase activity. To test this prediction, mutant proteins bearing point mutations within the domain responsible for the ribonucleic activity were created. We observed that RNase R mutants devoid of ribonuclease activity retained their ability to complement the cold shock function of CsdA. Purified ribonuclease-negative mutant proteins also exhibited helicase activity in vitro. RNase R has three cold shock domains that are known to possess unwinding activity in other proteins (most notably, CspA). Analysis of RNase R mutants revealed that only one cold shock domain of RNase R, the CSD2 domain, is important for its helicase activity. In vivo studies showed that this domain is not essential for the ribonuclease activity of RNase R.

PNPase.

Regulation of expression. PNPase is encoded by the pnp gene.13,14 Its expression is post-transcriptionally autoregulated at the level of both translation and mRNA stability.104 It has been shown that PNPase binds to the 5′ end of RNase III-processed pnp transcript, leading to the inhibition of translation and channeling of pnp mRNA into degradation pathway.105–107 During the cold acclimation phase this regulation is temporarily relieved leading to stabilization of the pnp mRNA, making it extremely abundant. CspA-dependent transcription antitermination at intercistronic intrinsic terminators also plays a role in transient induction of promoter-distal pnp gene. It has also been shown that suppression of Rho-dependent transcription termination within the pnp gene and its restoration by PNPase constitutes an autogenous regulatory circuit that modulates pnp expression during cold acclimation phase. The 5′-untranslated region of pnp mRNA plays a role in this phenomenon.108

Structure-function. PNPase is a homotrimer. Each 711 amino acid monomer consists of two RNA binding domains, KH and S1, located at the C-terminal portion of the polypeptide. The duplicated core of the protein is responsible for the catalytic function and homotrimerization.109–111 Both first and second core domains mediate the catalysis of the phosphorolytic reaction. Both the phosphorolytic and RNA binding activities are required for autogenous regulation of PNPase.106 It has also been shown that the KH and S1 domains of PNPase are important for its proper degradosome activity at low temperature. Note that at low temperatures, the stem-loop structures present in the target mRNAs are more stable.112

PNPase is one of the main exoribonucleases in the cell;113 it promotes processive degradation of RNA. PNPase is essential at low temperature for cell growth, although its exact role is not fully known.114,115 Recent data suggest that the ribonuclease activity of PNPase is critical for its cold shock function.98 By selectively degrading csp RNAs PNPase decreases the production of CspA and its cold-induced homologs at the end of the acclimation phase. In a pnp mutant, expression of CspA homologs was significantly prolonged upon cold shock.87,116 PNPase has been shown to be associated with endonuclease RNase E and other proteins in the RNA degradosome.117–119 In vitro, PNPase catalyzes phosphorolytic degradation of RNA, releasing nucleoside 5′-diphosphate from the 3′ end of the substrate and also carries out the reverse reaction of nucleoside 5′-diphosphate polymerization with the release of phosphate;120 both the phosphorolytic and polymerization activities are allosterically inhibited by ATP.121 The polymerization activity of PNPase is responsible for residual RNA tailing observed in E. coli mutants devoid of the main polyadenylating enzyme PAP I.122,123 PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge.

RNA Metabolism at Low Temperature

The RNA metabolism at low temperature may be different than that at 37°C and may require (i) the assistance of proteins that destabilize RNA secondary structures that are stabilized upon temperature downshift, making them accessible to ribonucleases and (ii) cold-inducible ribonucleases that can carry out RNA degradation efficiently and selectively to allow cell growth at low temperature. Note that both CsdA and PNPase are essential only at low temperature. Interestingly, these two proteins seem to function independently of each other as they cannot complement each other's functions. RNase R, in spite of being an 3′–5′ exoribonuclease like PNPase, cannot complement its cold shock function, but can complement that of CsdA by virtue of its unwinding activity. This implies that these two groups of proteins have distinct RNA targets. A detailed, comparative analysis of the RNA targets of these proteins should elaborate on how these proteins affect the RNA metabolism at low temperature.

Concluding Remarks

Stabilization of the secondary structures in RNA affects transcription of certain genes and also renders some RNAs inaccessible to degradation by ribonucleases. This results in inhibition or extensive slowing down of cell growth at low temperature. Cells produce two main groups of cold shock proteins, which modulate the RNA secondary structures and thus play important role(s) in the cold-shock adaptation of cells. It is interesting why cells need so many different proteins to facilitate RNA degradation at low temperature. While at least some of the proteins discussed here can partially complement for each others absence at cold shock, the complementation is never complete (and sometimes is totally absent). The result suggest multiple and only partly overlapping targets (presumably, nascent or already transcribed RNAs) that cold shock proteins act upon. Elucidation of these targets and determining the modes of action of various cold shock proteins on each target will be necessary for complete understanding of cellular adaptation to low temperatures.

Figures and Tables

Figure 1 A schematic representation of modulation of RNA by the cold shock proteins of E. coli.

Acknowledgements

This work was supported by NIH RO3 Grant 76900 to S.P. and G.M. “RO1 64530, Russian Academy of Sciences Presidium Molecular and Cell Biology Program grant and Federal Program “Scientific and pedagogical cadre of innovative Russia” 2009–2013,” State contract 02.740.11.0771 to K.S.