Rotavirus RNA chaperone mediates global transcriptome-wide increase in RNA backbone flexibility

Due to genome segmentation, rotaviruses must co-package a set of eleven distinct genomic RNAs. The packaging is mediated by virus-encoded RNA chaperones, such as the rotavirus (RV) NSP2 protein. While the activities of distinct viral RNA chaperones are well studied on synthetic RNA substrates, little is known about their global effect on the entire viral transcriptome. Here we used Selective 2′-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to systematically examine the secondary structure of the RV transcriptome composed of eleven distinct transcripts in the absence and presence of increasing concentrations of RV NSP2. Surprisingly, SHAPE-MaP data reveals that despite the well-documented helix-unwinding activity of NSP2 in vitro, its incubation with cognate RV transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of the per nucleotide mutation rate measured by mutational profiling, from which SHAPE reactivities are derived, reveals a global five-fold rate increase in the presence of molar excess of NSP2. We demonstrate that the standard normalization procedure used in deriving SHAPE reactivities from mutation rates can mask an important global effect of an RNA chaperone activity. Further analysis of the mutation rate in the context of structural classification reveals a larger effect on stems rather than loop elements. Together, these data provide the first experimentally derived secondary structure model of the RV transcriptome and reveal that NSP2 acts by globally increasing RNA backbone flexibility in a concentration-dependent manner, consistent with its promiscuous RNA-binding nature.

The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.

Structural basis of rotavirus RNA chaperone displacement and RNA annealing

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA–RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo–electron microscopy reconstructions of an NSP2–RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.

Differential Chromosome- and Plasmid-Borne Resistance of Escherichia coli hfq Mutants to High Concentrations of Various Antibiotics

The Hfq protein is a bacterial RNA chaperone, involved in many molecular interactions, including control of actions of various small RNA regulatory molecules. We found that the presence of Hfq was required for survival of plasmid-containing Escherichia coli cells against high concentrations of chloramphenicol (plasmid p27cmr), tetracycline (pSC101, pBR322) and ampicillin (pBR322), as hfq+ strains were more resistant to these antibiotics than the hfq-null mutant. In striking contrast, production of Hfq resulted in low resistance to high concentrations of kanamycin when the antibiotic-resistance marker was chromosome-borne, with deletion of hfq resulting in increasing bacterial survival. These results were observed both in solid and liquid medium, suggesting that antibiotic resistance is an intrinsic feature of these strains rather than a consequence of adaptation. Despite its major role as RNA chaperone, which also affects mRNA stability, Hfq was not found to significantly affect kan and tet mRNAs turnover. Nevertheless, kan mRNA steady-state levels were higher in the hfq-null mutant compared to the hfq+ strain, suggesting that Hfq can act as a repressor of kan expression.This observation does correlate with the enhanced resistance to high levels of kanamycin observed in the hfq-null mutant. Furthermore, dependency on Hfq for resistance to high doses of tetracycline was found to depend on plasmid copy number, which was only observed when the resistance marker was expressed from a low copy plasmid (pSC101) but not from a medium copy plasmid (pBR322). This suggests that Hfq may influence survival against high doses of antibiotics through mechanisms that remain to be determined. Studies with pBR322Δrom may also suggest an interplay between Hfq and Rom in the regulation of ColE1-like plasmid replication. Results of experiments with a mutant devoid of the part of the hfq gene coding for the C-terminal region of Hfq suggested that this region, as well as the N-terminal region, may be involved in the regulation of expression of antibiotic resistance in E. coli independently.

Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

The Multiple Regulatory Relationship Between RNA-Chaperone Hfq and the Second Messenger c-di-GMP

RNA chaperone protein Hfq is an important post-transcriptional regulator in bacteria, while c-di-GMP is a second messenger signaling molecule widely distributed in bacteria. Both factors have been found to play key roles in post-transcriptional regulation and signal transduction pathways, respectively. Intriguingly, the two factors show some common aspects in the regulation of certain physiological functions such as bacterial motility, biofilm formation, pathogenicity and so on. Therefore, there may be regulatory relationship between Hfq and c-di-GMP. For example, Hfq can directly regulate the activity of c-di-GMP metabolic enzymes or alter the c-di-GMP level through other systems, while c-di-GMP can indirectly enhance or inhibit the hfq gene expression through intermediate factors. In this article, after briefly introducing the Hfq and c-di-GMP regulatory systems, we will focus on the direct and indirect regulation reported between Hfq and c-di-GMP, aiming to compare and link the two regulatory systems to further study the complicated physiological and metabolic systems of bacteria, and to lay a solid foundation for drawing a more complete global regulatory network.

The RNA Chaperone Hfq Participates in Persistence to Multiple Antibiotics in the Fish Pathogen Yersinia ruckeri

Yersinia ruckeri causes outbreaks of enteric redmouth disease in salmon aquaculture all over the world. The transient antibiotic tolerance exhibited by bacterial persisters is commonly thought to be responsible for outbreaks; however, the molecular factors underlying this behavior have not been explored in Y. ruckeri. In this study, we investigated the participation of the RNA chaperone Hfq from Y. ruckeri in antibiotic persistence. Cultures of the hfq-knockout mutant (Δhfq) exhibited faster replication, increased ATP levels and a more reductive environment than the wild type. The growth curves of bacteria exposed to sublethal concentrations of ampicillin, oxolinic acid, ciprofloxacin and polymyxin B revealed a greater susceptibility for the Δhfq strain. The time-kill curves of bacteria treated with the antibiotics mentioned above and florfenicol, using inoculums from exponential, stationary and biofilm cultures, demonstrated that the Δhfq strain has significant defects in persister cells production. To shed more light on the role of Hfq in antibiotic persistence, we analyzed its dependence on the (p)ppGpp synthetase RelA by determining the persister cells production in the absence of the relA gene. The ΔrelA and ΔrelAΔhfq strains displayed similar defects in persister cells formation, but higher than Δhfq strain. Similarly, stationary cultures of the ΔrelA and ΔrelAΔhfq strains exhibited comparable levels of ATP but higher than that of the Δhfq strain, indicating that relA is epistatic over hfq. Taken together, our findings provide valuable information on antibiotic persistence in Y. ruckeri, shedding light on the participation of Hfq in the persistence phenomenon.

Conservation of Archaeal C/D Box sRNA-Guided RNA Modifications

Post-transcriptional modifications fulfill many important roles during ribosomal RNA maturation in all three domains of life. Ribose 2'-O-methylations constitute the most abundant chemical rRNA modification and are, for example, involved in RNA folding and stabilization. In archaea, these modification sites are determined by variable sets of C/D box sRNAs that guide the activity of the rRNA 2'-O-methyltransferase fibrillarin. Each C/D box sRNA contains two guide sequences that can act in coordination to bridge rRNA sequences. Here, we will review the landscape of archaeal C/D box sRNA genes and their target sites. One focus is placed on the apparent accelerated evolution of guide sequences and the varied pairing of the two individual guides, which results in different rRNA modification patterns and RNA chaperone activities.