Dissecting RNA chaperone activity

Many RNA-binding proteins help RNAs to fold via their RNA chaperone activity. This term has been used widely without accounting for the diversity of the observed reactions, which include complex events like restructuring of misfolded catalytic RNAs, promoting the assembly of RNA-protein complexes, and mediating RNA-RNA interactions. Proteins display very diverse activities depending on the assays used to measure RNA chaperone activity. To classify proteins with this activity, we compared three exemplary proteins from E. coli, host factor Hfq, ribosomal protein S1, and the histone-like protein StpA for their abilities to promote two simple reactions, RNA annealing and strand displacement. The results of a FRET-based assay show that S1 promotes only RNA strand displacement while Hfq solely enhances RNA annealing. StpA, in contrast, is active in both reactions. To test whether the two activities can be assigned to different domains of the bipartite-structured StpA, we assayed the purified N- and C- terminal domains separately. While both domains are unable to promote RNA annealing, we can attribute the RNA strand displacement activity of StpA to the C-terminal domain. Correlating with their RNA annealing activities, only Hfq and full-length StpA display simultaneous binding of two RNAs, suggesting a matchmaker-like model for this activity. For StpA, this "RNA crowding" requires protein-protein interactions, since a dimerization-deficient StpA mutant lost the ability to bind and anneal two RNAs. These results underline the difference between the two reaction types, making it necessary to distinguish and classify proteins according to their specific RNA chaperone activities.     

RNA chaperone activity and RNA-binding properties of the E. coli protein StpA

The E. coli protein StpA has RNA annealing and strand displacement activities and it promotes folding of RNAs by loosening their structures. To understand the mode of action of StpA, we analysed the relationship of its RNA chaperone activity to its RNA-binding properties. For acceleration of annealing of two short RNAs, StpA binds both molecules simultaneously, showing that annealing is promoted by crowding. StpA binds weakly to RNA with a preference for unstructured molecules. Binding of StpA to RNA is strongly dependent on the ionic strength, suggesting that the interactions are mainly electrostatic. A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity. This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA. We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.     

Study of E. coli Hfq’s RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents

Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other.     

RNA binding property and RNA chaperone activity of dengue virus core protein and other viral RNA-interacting proteins

In this study we showed that the dengue virus (DENV) core protein forms a dimer with an α-helix-rich structure, binds RNA and facilitates the strand annealing process. To assess the RNA chaperone activity of this core protein and other dengue viral RNA-interacting proteins, such as NS3 helicase and NS5 proteins, we engineered cis- and trans-cleavage hammerhead ribozyme constructs carrying DENV genomic RNA elements. Our results indicate that DENV core protein facilitates typical hammerhead structure formation by acting as an RNA chaperone and DENV NS5 has a weak RNA chaperone activity, while DENV NS3 helicase failed to refold RNA with a complex secondary structure.     

Effect of salt and RNA structure on annealing and strand displacement by Hfq

The Sm-like protein Hfq promotes the association of small antisense RNAs (sRNAs) with their mRNA targets, but the mechanism of Hfq''s RNA chaperone activity is unknown. To investigate RNA annealing and strand displacement by Hfq, we used oligonucleotides that mimic functional sequences within DsrA sRNA and the complementary rpoS mRNA. Hfq accelerated at least 100-fold the annealing of a fluorescently labeled molecular beacon to a 16-nt RNA. The rate of strand exchange between the oligonucleotides increased 80-fold. Therefore, Hfq is very active in both helix formation and exchange. However, high concentrations of Hfq destabilize the duplex by preferentially binding the single-stranded RNA. RNA binding and annealing were completely inhibited by 0.5 M salt. The target site in DsrA sRNA was 1000-fold less accessible to the molecular beacon than an unstructured oligonucleotide, and Hfq accelerated annealing with DsrA only 2-fold. These and other results are consistent with recycling of Hfq during the annealing reaction, and suggest that the net reaction depends on the relative interaction of Hfq with the products and substrates.     

RNA chaperone activity of protein components of human Ro RNPs

Ro ribonucleoprotein (RNP) complexes are composed of one molecule of a small noncoding cytoplasmic RNA, termed Y RNA, and the two proteins Ro60 and La. Additional proteins such as hnRNP I, hnRNP K, or nucleolin have recently been shown to be associated with subpopulations of Y RNAs. Ro RNPs appear to be localized in the cytoplasm of all higher eukaryotic cells but their functions have remained elusive. To shed light on possible functions of Ro RNPs, we tested protein components of these complexes for RNA chaperone properties employing two in vitro chaperone assays and additionally an in vivo chaperone assay. In these assays the splicing activity of a group I intron is measured. La showed pronounced RNA chaperone activity in the cis-splicing assay in vitro and also in vivo, whereas no activity was seen in the trans-splicing assay in vitro. Both hnRNP I and hnRNP K exhibited strong chaperone activity in the two in vitro assays, however, proved to be cytotoxic in the in vivo assay. No chaperone activity was observed for Ro60 in vitro and a moderate activity was detected in vivo. In vitro chaperone activities of La and hnRNP I were completely inhibited upon binding of Y RNA. Taken together, these data suggest that the Ro RNP components La, hnRNP K, and hnRNP I possess RNA chaperone activity, while Ro60-Y RNA complexes might function as transporters, bringing other Y RNA binding proteins to their specific targets.     

Identification of the RNA chaperone activity of recombinant human tumor necrosis factor alpha in vitro

RNA chaperones are defined as proteins that aid in the process of RNA folding by processing misfolding or by resolving misfolded structures. Although RNA chaperones are ubiquitous and abundant in all living organisms and viruses, there are no any reports that a cytokine has such RNA chaperone activity. Here, we demonstrate for the first time that recombinant human tumor necrosis factor alpha (rhTNF-alpha), a well-known cytokine, has RNA chaperone activity in vitro. rhTNF-alpha binds random 68 nt RNAs strongly at the minimal concentration of 10 microM with a broad sequence specificity. Our results also show that rhTNF-alpha facilitates annealing and strand exchange, and promotes the cleavage of a 17-nucleotide substrate S by hammerhead ribozyme HH16. The role of TNF-alpha as an RNA chaperone in vivo is not clear, but we propose that TNF-alpha may play an important role as an RNA chaperone during the process of some infectious and inflammatory diseases.     

Dengue virus 2 capsid protein chaperones the strand displacement of 5′-3′ cyclization sequences

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5′-3′ panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5′-3′ panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.     

RNA structural rearrangement via unwinding and annealing by the cyanobacterial RNA helicase, CrhR

Rearrangement of RNA secondary structure is crucial for numerous biological processes. RNA helicases participate in these rearrangements through the unwinding of duplex RNA. We report here that the redox-regulated cyanobacterial RNA helicase, CrhR, is a bona fide RNA helicase possessing both RNA-stimulated ATPase and bidirectional ATP-stimulated RNA helicase activity. The processivity of the unwinding reaction appears to be low, because RNA substrates containing duplex regions of 41 bp are not unwound. CrhR also catalyzes the annealing of complementary RNA into intermolecular duplexes. Uniquely and in contrast to other proteins that perform annealing, the CrhR-catalyzed reactions require ATP hydrolysis. Through a combination of the unwinding and annealing activities, CrhR also catalyzes RNA strand exchange resulting in the formation of RNA secondary structures that are too stable to be resolved by helicase activity. RNA strand exchange most probably occurs through the CrhR-dependent formation and resolution of an RNA branch migration structure. Demonstration that another cyanobacterial RNA helicase, CrhC, does not catalyze annealing indicates that this activity is not a general biochemical characteristic of RNA helicases. Biochemically, CrhR resembles RecA and related proteins that catalyze strand exchange and branch migration on DNA substrates, a characteristic that is reflected in the recently reported structural similarities between these proteins. The data indicate the potential for CrhR to catalyze dynamic RNA secondary structure rearrangements through a combination of RNA helicase and annealing activities.     

Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72

RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion. Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps. The two proteins resemble each other also with regard to other biochemical properties. We have found that both proteins exhibit an RNA annealing in addition to their helicase activity. By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities. RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis. The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.     

RNA chaperone activity of translation initiation factor IF1

Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.     

RNA annealing activities in HeLa nuclei.

RNA-RNA base pairing plays a critical role in the interactions between pre-mRNAs and trans-acting factors during the processing of pre-mRNAs (hnRNAs) into mRNAs, and it is likely that specific factors are required to promote the annealing of RNAs. To identify particular nuclear components that have such activity, we fractionated HeLa nucleoplasm and assayed for activity which promoted the hybridization of a pre-mRNA with an antisense RNA probe complementary to 60 nucleotides (nt) encompassing the 3' splice site. At least nine major RNA annealing activities were identified and, surprisingly, eight of these copurified partially or to homogeneity with known hnRNP proteins. The activities of three of these proteins, hnRNP A1, C1 and U, were confirmed using purified recombinant proteins. Moreover, we found that the RNA binding domain alone of hnRNP C1/C2 had significant activity, indicating that this RNA annealing may result, at least partly, from chaperone activity: a direct modulation of RNA conformation by hnRNP proteins. The finding that hnRNP proteins have strong RNA annealing activity indicates that they can profoundly affect the interactions of pre-mRNAs with trans-acting factors and suggests this to be an important function of hnRNP proteins in the processing of pre-mRNAs.     

The double-stranded RNA-binding protein X1rbpa promotes RNA strand annealing.

RNA-annealing activity is a common feature of several RNA-binding proteins. The Xenopus RNA-binding protein X1rbpa is composed of three tandemly arranged double-stranded RNA-binding domains (dsRBDs) but lacks any other catalytic or functional domains, therefore making the assessment of biological functions of this protein rather difficult. Here we show that full-length X1rbpa but also isolated dsRBDs from this protein can facilitate RNA strand annealing. RNA annealing can be efficiently inhibited by heparin. However, dsRBDs with a neutral pI still promote strand annealing, suggesting that charged residues within the dsRBD are important for strand annealing. Additionally, mutant versions of the dsRBD, unable to bind dsRNA in northwestern assays, were tested. Of these, some show RNA-annealing activity while others fail to do so, indicating that RNA annealing and dsRNA binding are separable functions. Our data, together with the previously reported association of the protein with most cellular RNAs, suggests an RNA chaperone-like function of X1rbpa.     

RNA chaperone activity of large ribosomal subunit proteins from Escherichia coli

The ribosome is a highly dynamic ribonucleoprotein machine. During assembly and during translation the ribosomal RNAs must routinely be prevented from falling into kinetic folding traps. Stable occupation of these trapped states may be prevented by proteins with RNA chaperone activity. Here, ribosomal proteins from the large (50S) ribosome subunit of Escherichia coli were tested for RNA chaperone activity in an in vitro trans splicing assay. Nearly a third of the 34 large ribosomal subunit proteins displayed RNA chaperone activity. We discuss a possible role of this function during ribosome assembly and during translation.     

RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus.

Most positive strand RNA viruses infecting plants and animals encode proteins containing the so-called nucleotide binding motif (NTBM) (1) in their amino acid sequences (2). As suggested from the high level of sequence similarity of these viral proteins with the recently described superfamilies of helicase-like proteins (3-5), the NTBM-containing cylindrical inclusion (CI) protein from plum pox virus (PPV), which belongs to the potyvirus group of positive strand RNA viruses, is shown to be able to unwind RNA duplexes. This activity was found to be dependent on the hydrolysis of NTP to NDP and Pi, and thus it can be considered as an RNA helicase activity. In the in vitro assay used, the PPV CI protein was only able to unwind double strand RNA substrates with 3' single strand overhangs. This result indicates that the helicase activity of the PPV CI protein functions in the 3' to 5' direction (6). To our knowledge, this is the first report on a helicase activity associated with a protein encoded by an RNA virus.     

The RNA Chaperone and Protein Chaperone Activity of Arabidopsis Glycine-Rich RNA-Binding Protein 4 and 7 is Determined by the Propensity for the Formation of High Molecular Weight Complexes

RNA chaperones and protein chaperones are cellular proteins that can aid the correct folding of target RNAs and proteins, respectively. Although many proteins possessing RNA chaperone or protein chaperone activity have been demonstrated in diverse organisms, report evaluating the RNA chaperone and protein chaperone activity of a given protein is severely limited. Here, two glycine-rich RNA-binding proteins in Arabidopsis thaliana (AtGRPs), AtGRP7 exhibiting RNA chaperone activity and AtGRP4 exhibiting no RNA chaperone activity, were investigated for their protein chaperone activity. The heat-induced thermal aggregation of a substrate protein was significantly decreased with the addition of AtGRP4 depending on protein concentration, whereas the thermal aggregation of a substrate protein was further increased with the addition of AtGRP7, demonstrating that AtGRP4 but not AtGRP7 possesses protein chaperone activity. Size exclusion chromatography and electron microscopy analyses revealed that the formation of high molecular weight (HMW) complexes is closely related to the protein chaperone activity of AtGRP4. Importantly, the additional 25 amino acids at the N-terminus of AtGRP4 are crucial for HMW complex formation and protein chaperone activity. Taken together, these results show that the formation of HMW complexes is important for determining the RNA chaperone and protein chaperone activity of AtGRP4 and AtGRP7.     

The nonstructural protein 2C of Coxsackie B virus has RNA helicase and chaperoning activities

RNA-remodeling proteins, including RNA helicases and chaperones, play vital roles in the remodeling of structured RNAs. During viral replication, viruses require RNA-remodeling proteins to facilitate proper folding and/or re-folding the viral RNA elements. Coxsackieviruses B3 (CVB3) and Coxsackieviruses B5 (CVB5), belonging to the genus Enterovirus in the family Picornaviridae, have been reported to cause various infectious diseases such as hand-foot-and-mouth disease, aseptic meningitis, and viral myocarditis. However, little is known about whether CVB3 and CVB5 encode any RNA remodeling proteins. In this study, we showed that 2C proteins of CVB3 and CVB5 contained the conserved SF3 helicase A, B, and C motifs, and functioned not only as RNA helicase that unwound RNA helix bidirectionally in an NTP-dependent manner, but also as RNA chaperone that remodeled structured RNAs and facilitated RNA strand annealing independently of NTP. In addition, we determined that the NTPase activity and RNA helicase activity of 2C proteins of CVB3 and CVB5 were dependent on the presence of divalent metallic ions. Our findings demonstrate that 2C proteins of CVBs possess RNA-remodeling activity and underline the functional importance of 2C protein in the life cycle of CVBs.     

RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae

RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning—possibly mediated by intrinsically disordered protein segments—is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.     

Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2C^ATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2C^ATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2C^ATPase facilitated EV71 RNA synthesis in vitro; when 2C^ATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2C^ATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2C^ATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2C^ATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.