Unwinding activity of cold shock proteins and RNA metabolism

Abstract

Temperature downshift from 37°C to 15°C results in the exertion of cold shock response in Escherichia coli, which induces cold shock proteins, such as CsdA. Previously, we showed that the helicase activity of CsdA is critical for its function in the cold acclimation of cells and its primary role is mRNA degradation. Only RhlE (helicase), CspA (RNA chaperone) and RNase R (exoribonuclease) were found to complement the cold shock function of CsdA. RNase R has two independent activities, helicase and ribonuclease, only helicase being essential for the functional complementation of CsdA. Here, we discuss the significance of above findings as these emphasize the importance of the unwinding activity of cold-shock-inducible proteins in the RNA metabolism at low temperature, which may be different than that at 37°C. It requires assistance of proteins to destabilize the secondary structures in mRNAs that are stabilized upon temperature downshift, hindering the activity of ribonucleases.

In E. coli, the cold-shock response is exerted when a culture growing exponentially at 37°C is shifted to 15°C. Upon temperature downshift, there is a lag period of growth termed acclimation phase, in which cellular synthesis of most of the proteins is inhibited in contrast to that of a select group of proteins, termed cold-shock proteins. Cold shock proteins presumably help the cells counteract various detrimental cellular changes caused by the temperature downshift. This is followed by resumption of the cell growth with restoration of synthesis of normal cellular proteins and decrease in the rate of synthesis of cold-shock proteins. The cold-shock proteins include CspA¹ and its homologues such as CspB,² CspG³ and CspI,⁴ RNA helicase CsdA,⁵ DNA gyrase,⁶ histone-like protein H-NS,⁷ initiation factor IF2,⁸ transcription factor NusA,⁹ polynucleotide phosphorylase,¹⁰ ribosome-binding factor RbfA¹¹ and RecA.¹²

Role of CsdA, a RNA Helicase, in Cold Shock Response

E. coli contains five DEAD-box genes, csdA (formerly called deaD), dbpA, rhlB, rhlE and srmB. CsdA is an RNA helicase belonging to the family of proteins which is conserved from bacteria to humans.¹³ Along with the related DExD/H-box proteins, these RNA helicases play important roles in many cellular processes such as processing, transport or degradation of RNA or ribosome biogenesis.^14,15 CsdA is essential only at low temperature^16,17 and has been suggested to have multiple functions as (1) the biogenesis of the small ribosomal subunit^5,18 and the 50S ribosomal subunits.¹⁶ Deletion of the csdA gene leads to a shortage of free 50S subunits and accumulation of a 40S-like particle,¹⁶ (2) promotion of translation initiation of structured mRNAs,¹⁹ (3) stabilization and degradation of mRNAs,^20–22 (4) it accumulates during the initial stages of cold acclimation and assembles into degradosomes with RNase E.²² CsdA may also be involved in the efficient and selective degradation of Csp mRNAs by unwinding the mRNA secondary structure and thereby helping the processing activity of PNPase.²⁰ Taken together, these data suggested that CsdA has multiple functions in several important physiological processes, however, its exact role in its essential cold-shock function was not clear and no protein was shown to complement its function in vivo. We therefore undertook studies to elucidate the cold-shock-acclimation role of CsdA and demonstrated that mRNA decay may be its primary role and its helicase activity is crucial for promoting degradation of mRNAs stabilized at low temperature.²³ The correlation between the helicase activity of CsdA and stability of mRNAs of cold-inducible genes was shown at 15°C using a target mRNA, which was significantly stabilized in the ΔcsdA cells; an effect counteracted by overexpression of wild-type CsdA, but not by a helicase-deficient mutant of CsdA.

Is CsdA Unique Helicase in E. coli?

The key observations about the E. coli DEAD-box RNA helicases¹⁴ include (1) these proteins do not show strong helicase activity in vitro and can unwind only a few base pairs at each cycle. Turner et al. reported that CsdA shows significantly less helicase activity in the in vitro assays at 15°C, (2) with the exception of DbpA, these proteins do not show high degree of specificity in vitro, although may show specificity for RNA substrates in vivo presumably by interacting with other proteins which target them to specific substrates, (3) despite being conserved during evolution they are not essential at 37°C and rhlB, rhlE and dbpA can be individually deleted without a noticeable effect on growth, whereas the deletion of srmB and csdA leads to a growth defect only at low temperature, (4) the double mutants (ΔcsdAΔsrmB, ΔcsdAΔrhlE, ΔcsdAΔdbpA, ΔrhlEΔdbpA, ΔrhlEΔsrmB and ΔsrmBΔdbpA) did not show any effect on growth, (5) these are not generally interchangeable in vivo, except for RhlE, which can compliment the cold shock function of CsdA as described below. It was suggested that this is presumably due to their different C-terminal extensions flanking the DEAD-core sequence. These extensions vary in size (from ∼70 to 290 amino acids) and in sequence. The C-terminal extensions may facilitate the interaction of these proteins with their specific RNA substrates or interacting partner proteins.

Our in vivo genetic screening of an E. coli csdA deletion strain and results from other research groups revealed that another DEAD-box RNA helicase, RhlE can compensate for CsdA function at low temperature.^14,23,25 We further observed that although not detected in our genetic screen, two cold-shock-inducible proteins, an RNA chaperone, CspA, and an exonuclease, RNase R can also complement cold-shock function of CsdA. We also observed that absence of CsdA and RNase R results in increased sensitivity of the cells to moderate temperature downshifts.

RNase R has Dual Activities, Helicase and Ribonuclease

RNase R, RNase II and PNPase are the three major 3′-to-5′ processing exoribonucleases primarily involved in the RNA metabolism in E. coli, out of which RNase R²⁶ and PNPase^27,28 are cold shock inducible. Despite the fact that similar to RNase R, PNPase is presumably the universal degrader of structured RNA in vivo,^29,30 it could not complement the helicase activity of CsdA.²³ This suggested 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 it may also possess helicase activity. It is also interesting that RNase II and RNase R belong to the RNR family and exhibit approximately 60% similarity in their secondary structures, but RNase II cannot complement cold shock function of CsdA.

RNase R contains a central nuclease domain, two cold-shock (CSD) domains near the N-terminus region of the protein, an S1 domain and a highly basic region near the C-terminus of the protein.³¹ We carried out in vivo domain analysis of RNase R and showed that it has helicase activity independent of its ribonuclease activity and this activity is essential for complementation of the cold shock function of CsdA. Thus, the mutant RNase R proteins completely lacking the ribonuclease activity retained the helicase activity and the ability to complement the cold shock function of CsdA. We also observed that in vivo, the presence of the CSD2 domain seems to be critical for the helicase activity of RNase R and either the CSD1 or the S1 domain is required for optimal activity. The in vitro experiments with the purified wild-type and mutant RNase R proteins too showed that RNase R possesses helicase activity that is independent of its ribonuclease activity. The results emphasized that RNase R is unique in that it is the only ribonucleases in E. coli that complements the cold sensitive phenotype of the csdA deletion cells. Further studies, especially structural analysis are required to determine why the CSD2 domain is critical for the helicase activity of RNase R. Our results showed that the CSD2 is not critical for the ribonuclease activity of RNase R and another group showed that the nuclease domain of RNase R was sufficient for the digestion of structured RNAs.³¹

Proteins with RNA Unwinding Activity and RNA Metabolism at Low Temperature

As mentioned above, CsdA is a multi-functional protein; two of its activities are the subject of intensive study, its role in mRNA decay and ribosome biogenesis. It seems that the unwinding (helicase) activity of CsdA may be important for both of these functions. As mentioned above, the helicase activity of CsdA is critical for its important cold shock function, namely mRNA decay and helicase-negative mutants of CsdA failed to complement the cold sensitive phenotype of the csdA deletion cells and the target mRNAs were stabilized in the cells carrying these mutants.²³ It was suggested that CsdA may help 50S assembly by modulating RNA or RNP structures and its unwinding activity may be required to facilitate structural transitions within the RNA and may also allow proper binding of r-protein(s).¹⁴ Alternatively, CsdA may prevent and/or resolve misfolding, which may prove to be useful to provide assistance to rRNA to reach its active conformation the rRNA, which may otherwise be trapped in incorrect structures.

In the view of the above observations, it is interesting that in addition to the complementation of cold shock function of CsdA by RhlE, an RNA chaperone, CspA and an exoribonuclease, RNase R are also able to complement CsdA at low temperature, albeit somewhat weakly.²³ It is also interesting that similar to CsdA, both CspA¹ and RNase R²⁶ are induced by cold shock. There are some distinct similarities and differences in the RNA unwinding activities and functions of these three proteins, (1) CsdA, RNase R and CspA can not act on blunt ended substrates; CsdA and CspA can act on substrates with both 3′ or 5′ extensions,^24,32 while RNase R requires presence of a 3′ overhang in its substrate, (2) CsdA and CspA exhibit low unwinding activity. Many DEAD-box RNA helicases have been shown to have extremely low processivity of unwinding.^33–35 These enzymes have been demonstrated to possess little or no unwinding on substrates longer than 12–15 bp. CspE, a prototype member of the E. coli CspA family, was shown to melt 9 bp duplexes, but failed to efficiently melt duplexes, which were 14 bp in length,³² (3) CspA has been shown to act as an RNA chaperone, which destabilizes the secondary structures in nucleic acids, while RNase R is unique among exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3′ overhang is present,³⁶ suggesting some unwinding of the secondary structures followed by degradation of RNAs may be involved, (4) RNase R also resembles CsdA in another aspect that in Pseudomonas syringae, it has been shown to interact with the endoribonuclease RNase E and an RNA helicase, suggesting that in this bacterium, it is preferred over PNPase as a component of degradosome.³⁷ Complementation of the cold shock function of CsdA, by RhlE is not surprising, however the fact that CspA, a protein structurally unrelated to CsdA can do so by virtue of its RNA chaperone activity emphasizes the importance of the unwinding activity of cold-shock-inducible proteins in the RNA metabolism at low temperature. This suggests that the low-temperature RNA metabolism is distinctly different than that at 37°C and requires assistance from proteins to destabilize the secondary structures in RNA that are stabilized upon temperature downshift, which may hinder the activity of ribonucleases (Fig. 1). It has been shown that RNAs can act as thermometer and respond to temperature downshift.^38–41 Thermal controlling of the folding of certain mRNAs results in (1) hindrance in translation, by virtue of controlling the accessibility of translation initiation signals that are captured in hairpin structures which are stabilized by the temperature downshift and (2) inaccessibility to RNases for degradation of mRNAs which now assume stabilized secondary structures.

Figure 1.

A schematic representation of unwinding by CsdA (and RNase R/CspA) and metabolism of RNA at low temperature.

The hypothesis that RNA unwinding proteins play a role in RNA metabolism at low temperature is also supported by previous in vitro experimental data showing that indeed CspA or its homologues can facilitate cleavage reactions by ribonucleases. The presence of double-stranded secondary structures in the RNA substrate can be a limiting factor for ribonucleases, especially those which act only on single-stranded RNAs. Certain cleavage sites in the RNA substrate used for in vitro reactions were thus not accessible to RNase T or RNase E as these were present in the double stranded region. Inclusion of CspA or its homologue, CspE, rendered these sites accessible to ribonucleases by melting the double-stranded regions in the RNA substrate.^42,43 We also successfully used purified CspA to determine the cleavage specificity of Mycobacterium tuberculosis MazF (MazF-mt6), an endoribonuclease, which is a toxin that can act only on single-stranded RNAs. We used an RNA substrate with extensive secondary structures for the in vitro assays. When the purified MazF-mt6 was incubated with the RNA substrate in the absence of CspA, no cleavage of the RNA was detected. When purified CspA was included in the reaction mixture, MazF-mt6 was able to cleave the RNA.⁴⁴

The fact that only these proteins, which can complement the CsdA function have unwinding activity supports this hypothesis. The question now is why only these proteins can complement the CsdA function at cold shock. Previously, it was reported that CsdA does not exhibit strong specificity for RNA substrates in vitro, but it was suggested that it may do so in vivo.¹⁴ CspA does not have high specificity for nucleic acid substrates.⁴² In the case of RNase R, as its helicase activity is executed independent of its ribonuclease activity, the RNA substrates for these activities may be different or it may share a common subset of mRNAs, which it can both unwind and degrade. While RNase R may have some preference for RNA substrates it can degrade,⁴⁵ no data is available at present as to the preferred targets for its unwinding activity. A comparative DNA microarray analysis of the targets of these three proteins at cold shock condition should further our knowledge of the RNA metabolism at low temperature and answer the intriguing questions such as, (1) why a protein with limited unwinding activity is critical for RNA metabolism at low temperature, (2) what contributes to the substrate specificity of CsdA for unwinding in vivo and (3) which structural components render the RNAs susceptible to the helicase activity of CsdA.