Alternatively spliced isoforms of AUF1 regulate a miRNA-mRNA interaction differentially through their YGG motif.

Proper base-pairing of a miRNA with its target mRNA is a key step in miRNA-mediated mRNA repression. RNA remodelling by RNA-binding proteins (RBPs) can improve access of miRNAs to their target mRNAs. The largest isoform p45 of the RBP AUF1 has previously been shown to remodel viral or AU-rich RNA elements. Here, we show that AUF1 is capable of directly promoting the binding of the miRNA let-7b to its target site within the 3'UTR of the POLR2D mRNA. Our data suggest this occurs in two ways. First, the helix-destabilizing RNA chaperone activity of AUF1 disrupts a stem-loop structure of the target mRNA and thus exposes the miRNA target site. Second, the RNA annealing activity of AUF1 drives hybridization of the miRNA and its target site within the mRNA. Interestingly, the RNA remodelling activities of AUF1 were found to be isoform-specific. AUF1 isoforms containing a YGG motif are competent RNA chaperones, whereas isoforms lacking the YGG motif are not. Overall, our study demonstrates that AUF1 has the ability to modulate a miRNA-target site interaction, thus revealing a new regulatory function for AUF1 proteins during post-transcriptional control of gene expression. Moreover, tests with other RBPs suggest the YGG motif acts as a key element of RNA chaperone activity.

The Host Factor AUF1 p45 Supports Flavivirus Propagation by Triggering the RNA Switch Required for Viral Genome Cyclization.

In previous studies, we showed that the cellular RNA-binding protein AUF1 supports the replication process of the flavivirus West Nile virus. Here we demonstrate that the protein also enables effective proliferation of dengue virus and Zika virus, indicating that AUF1 is a general flavivirus host factor. Further studies demonstrated that the AUF1 isoform p45 significantly stimulates the initiation of viral RNA replication and that the protein's RNA chaperone activity enhances the interactions of the viral 5'UAR and 3'UAR genome cyclization sequences. Most interestingly, we observed that AUF1 p45 destabilizes not only the 3'-terminal stem-loop (3'SL) but also 5'-terminal stem-loop B (SLB) of the viral genome. RNA structure analyses revealed that AUF1 p45 increases the accessibility of defined nucleotides within the 3'SL and SLB and, in this way, exposes both UAR cyclization elements. Conversely, AUF1 p45 does not modulate the fold of stem-loop A (SLA) at the immediate genomic 5' end, which is proposed to function as a promoter of the viral RNA-dependent RNA polymerase (RdRp). These findings suggest that AUF1 p45, by destabilizing specific stem-loop structures within the 5' and 3' ends of the flaviviral genome, assists genome cyclization and concurrently enables the RdRp to initiate RNA synthesis. Our study thus highlights the role of a cellular RNA-binding protein inducing a flaviviral RNA switch that is crucial for viral replication.IMPORTANCE The genus Flavivirus within the Flaviviridae family includes important human pathogens, such as dengue, West Nile, and Zika viruses. The initiation of replication of the flaviviral RNA genome requires a transformation from a linear to a cyclized form. This involves considerable structural reorganization of several RNA motifs at the genomic 5' and 3' ends. Specifically, it needs a melting of stem structures to expose complementary 5' and 3' cyclization elements to enable their annealing during cyclization. Here we show that a cellular RNA chaperone, AUF1 p45, which supports the replication of all three aforementioned flaviviruses, specifically rearranges stem structures at both ends of the viral genome and in this way permits 5'-3' interactions of cyclization elements. Thus, AUF1 p45 triggers the RNA switch in the flaviviral genome that is crucial for viral replication. These findings represent an important example of how cellular (host) factors promote the propagation of RNA viruses.

The RGG/RG motif of AUF1 isoform p45 is a key modulator of the protein's RNA chaperone and RNA annealing activities.

The RNA-binding protein AUF1 regulates post-transcriptional gene expression by affecting the steady state and translation levels of numerous target RNAs. Remodeling of RNA structures by the largest isoform AUF1 p45 was recently demonstrated in the context of replicating RNA viruses, and involves two RNA remodeling activities, i.e. an RNA chaperone and an RNA annealing activity. AUF1 contains two non-identical RNA recognition motifs (RRM) and one RGG/RG motif located in the C-terminus. In order to determine the functional significance of each motif to AUF1's RNA-binding and remodeling activities we performed a comprehensive mutagenesis study and characterized the wildtype AUF1, and several variants thereof. We demonstrate that each motif contributes to efficient RNA binding and remodeling by AUF1 indicating a tight cooperation of the RRMs and the RGG/RG motif. Interestingly, the data identify two distinct roles for the arginine residues of the RGG/RG motif for each RNA remodeling activity. First, arginine-mediated stacking interactions promote AUF1's helix-destabilizing RNA chaperone activity. Second, the electropositive character of the arginine residues is the major driving force for the RNA annealing activity. Thus, we provide the first evidence that arginine residues of an RGG/RG motif contribute to the mechanism of RNA annealing and RNA chaperoning.

NF90-NF45 is a selective RNA chaperone that rearranges viral and cellular riboswitches: biochemical analysis of a virus host factor activity.

The heterodimer NF90-NF45 is an RNA-binding protein complex that modulates the expression of various cellular mRNAs on the post-transcriptional level. Furthermore, it acts as a host factor that supports the replication of several RNA viruses. The molecular mechanisms underlying these activities have yet to be elucidated. Recently, we showed that the RNA-binding capabilities and binding specificity of NF90 considerably improves when it forms a complex with NF45. Here, we demonstrate that NF90 has a substrate-selective RNA chaperone activity (RCA) involving RNA annealing and strand displacement activities. The mechanism of the NF90-catalyzed RNA annealing was elucidated to comprise a combination of 'matchmaking' and compensation of repulsive charges, which finally results in the population of dsRNA products. Heterodimer formation with NF45 enhances 'matchmaking' of complementary ssRNAs and substantially increases the efficiency of NF90's RCA. During investigations of the relevance of the NF90-NF45 RCA, the complex was shown to stimulate the first step in the RNA replication process of hepatitis C virus (HCV) in vitro and to stabilize a regulatory element within the mRNA of vascular endothelial growth factor (VEGF) by protein-guided changes of the RNAs' structures. Thus, our study reveals how the intrinsic properties of an RNA-binding protein determine its biological activities.

Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45.

A prerequisite for the intracellular replication process of the Flavivirus West Nile virus (WNV) is the cyclization of the viral RNA genome, which enables the viral replicase to initiate RNA synthesis. Our earlier studies indicated that the p45 isoform of the cellular AU-rich element binding protein 1 (AUF1) has an RNA chaperone activity, which supports RNA cyclization and viral RNA synthesis by destabilizing a stem structure at the WNV RNA's 3'-end. Here we show that in mammalian cells, AUF1 p45 is consistently modified by arginine methylation of its C terminus. By a combination of different experimental approaches, we can demonstrate that the methyltransferase PRMT1 is necessary and sufficient for AUF1 p45 methylation and that PRMT1 is required for efficient WNV replication. Interestingly, in comparison to the nonmethylated AUF1 p45, the methylated AUF1 p45(aDMA) exhibits a significantly increased affinity to the WNV RNA termini. Further data also revealed that the RNA chaperone activity of AUF1 p45(aDMA) is improved and the methylated protein stimulates viral RNA synthesis considerably more efficiently than the nonmethylated AUF1 p45. In addition to its destabilizing RNA chaperone activity, we identified an RNA annealing activity of AUF1 p45, which is not affected by methylation. Arginine methylation of AUF1 p45 thus represents a specific determinant of its RNA chaperone activity while functioning as a WNV host factor. Our data suggest that the methylation modifies the conformation of AUF1 p45 and in this way affects its RNA binding and restructuring activities.

The properties of the RNA-binding protein NF90 are considerably modulated by complex formation with NF45.

Nuclear factor 90 (NF90) is an RNA-binding protein (RBP) that regulates post-transcriptionally the expression of various mRNAs. NF90 was recently shown to be capable of discriminating between different RNA substrates. This is mediated by an adaptive and co-operative interplay between three RNA-binding motifs (RBMs) in the protein's C-terminus. In many cell types, NF90 exists predominantly in a complex with NF45. Here, we compared the RNA-binding properties of the purified NF90 monomer and the NF90-NF45 heterodimer by biophysical and biochemical means, and demonstrate that the interaction with NF45 considerably affects the characteristics of NF90. Along with a thermodynamic stabilization, complex formation substantially improves the RNA-binding capacity of NF90 by modulating its binding mode and by enhancing its affinity for single- and double-stranded RNA substrates. Our data suggest that features of both the N- and C-termini of NF90 participate in the heterodimerization with NF45 and that the formation of NF90-NF45 changes the conformation of NF90's RBMs to a status in which the co-operative interplay of the RBMs is optimal. NF45 is considered to act as a conformational scaffold for NF90's RBMs, which alters the RNA-binding specificity of NF90. Accordingly, the monomeric NF90 and the NF90-NF45 heterodimer may exert different functions in the cell.

Coordinated Action of Two Double-Stranded RNA Binding Motifs and an RGG Motif Enables Nuclear Factor 90 To Flexibly Target Different RNA Substrates.

The mechanisms of how RNA binding proteins (RBP) bind to and distinguish different RNA molecules are yet uncertain. Here, we performed a comprehensive analysis of the RNA binding properties of multidomain RBP nuclear factor 90 (NF90) by investigating specifically the functional activities of two double-stranded RNA binding motifs (dsRBM) and an RGG motif in the protein's unstructured C-terminus. By comparison of the RNA binding affinities of several NF90 variants and their modes of binding to a set of defined RNA molecules, the activities of the motifs turned out to be very different. While dsRBM1 contributes little to RNA binding, dsRBM2 is essential for effective binding of double-stranded RNA. The protein's immediate C-terminus, including the RGG motif, is indispensable for interactions of the protein with single-stranded RNA, and the RGG motif decisively contributes to NF90's overall RNA binding properties. Conformational studies, which compared wild-type NF90 with a variant that contains a pseudophosphorylated residue in the RGG motif, suggest that the NF90 C-terminus is involved in conformational changes in the protein after RNA binding, with the RGG motif acting as a central regulatory element. In summary, our data propose a concerted action of all RNA binding motifs within the frame of the full-length protein, which may be controlled by regulation of the activity of the RGG motif, e.g., by phosphorylation. This multidomain interplay enables the RBP NF90 to discriminate RNA features by dynamic and adaptable interactions.

Homeologs of the Nicotiana benthamiana Antiviral ARGONAUTE1 Show Different Susceptibilities to microRNA168-Mediated Control.

The plant ARGONAUTE1 protein (AGO1) is a central functional component of the posttranscriptional regulation of gene expression and the RNA silencing-based antiviral defense. By genomic and molecular approaches, we here reveal the presence of two homeologs of the AGO1-like gene in Nicotiana benthamiana, NbAGO1-1H and NbAGO1-1L. Both homeologs retain the capacity to transcribe messenger RNAs (mRNAs), which mainly differ in one 18-nucleotide insertion/deletion (indel). The indel does not modify the frame of the open reading frame, and it is located eight nucleotides upstream of the target site of a microRNA, miR168, which is an important modulator of AGO1 expression. We demonstrate that there is a differential accumulation of the two NbAGO1-1 homeolog mRNAs at conditions where miR168 is up-regulated, such as during a tombusvirus infection. The data reported suggest that the indel affects the miR168-guided regulation of NbAGO1 mRNA. The two AGO1 homeologs show full functionality in reconstituted, catalytically active RNA-induced silencing complexes following the incorporation of small interfering RNAs. Virus-induced gene silencing experiments suggest a specific involvement of the NbAGO1 homeologs in symptom development. The results provide an example of the diversity of microRNA target regions in NbAGO1 homeolog genes, which has important implications for improving resilience measures of the plant during viral infections.

AUF1 p45 promotes West Nile virus replication by an RNA chaperone activity that supports cyclization of the viral genome.

UNLABELLED: A central aspect of current virology is to define the function of cellular proteins (host factors) that support the viral multiplication process. This study aimed at characterizing cellular proteins that assist the RNA replication process of the prevalent human pathogen West Nile virus (WNV). Using in vitro and cell-based approaches, we defined the p45 isoform of AU-rich element RNA-binding protein 1 (AUF1) as a host factor that enables efficient WNV replication. It was demonstrated that AUF1 p45 has an RNA chaperone activity, which aids the structural rearrangement and cyclization of the WNV RNA that is required by the viral replicase to initiate RNA replication. The obtained data suggest the RNA chaperone activity of AUF1 p45 is an important determinant of the WNV life cycle. IMPORTANCE: In this study, we identified a cellular protein, AUF1 (also known as heterogeneous ribonucleoprotein D [hnRNPD]), acting as a helper (host factor) of the multiplication process of the important human pathogen West Nile virus. Several different variants of AUF1 exist in the cell, and one variant, AUF1 p45, was shown to support viral replication most significantly. Interestingly, we obtained a set of experimental data indicating that a main function of AUF1 p45 is to modify and thus prepare the West Nile virus genome in such a way that the viral enzyme that generates progeny genomes is empowered to do this considerably more efficiently than in the absence of the host factor. The capability of AUF1 p45 to rearrange the West Nile virus genome was thus identified to be an important aspect of a West Nile virus infection.

RNA Remodeling by RNA Chaperones Monitored by RNA Structure Probing.

RNA structure probing enables the characterization of RNA secondary structures by established procedures such as the enzyme- or chemical-based detection of single- or double-stranded regions. A specific type of application involves the detection of changes of RNA structures and conformations that are induced by proteins with RNA chaperone activity. This chapter outlines a protocol to analyze RNA structures in vitro in the presence of an RNA-binding protein with RNA chaperone activity. For this purpose, we make use of the methylating agents dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT). DMS and CMCT specifically modify nucleotides that are not involved in base-pairing or tertiary structure hydrogen bonding and that are not protected by a ligand such as a protein. Modified bases are identified by primer extension. As an example, we describe how the RNA chaperone activity of an isoform of the RNA-binding protein AUF1 induces the flaviviral RNA switch required for viral genome cyclization and viral replication.This chapter includes comprehensive protocols for in vitro synthesis of RNA, 32P-5'-end labeling of DNA primers, primer extension, as well as the preparation and running of analytical gels. The described methodology should be applicable to any other RNA and protein of interest to identify protein-directed RNA remodeling.

Real-Time Fluorescence-Based Approaches to Disentangle Mechanisms of a Protein's RNA Chaperone Activity.

RNA-binding proteins with an RNA chaperone activity exert either one or both of the following catalytic activities: (1) RNA annealing, i.e., the protein supports intra- as well as intermolecular RNA-RNA interactions and (2) strand displacement, i.e., the protein mediates the exchange of individual strands of a preexisting RNA duplex. To discriminate and further characterize these activities, it requires defined assay systems. These are based on entirely or partially complementary RNA oligonucleotides that are labeled with fluorescent and/or quencher dyes. The non-catalyzed and the protein-supported associations of the RNA molecules are followed by a real-time fluorescence resonance energy transfer (FRET) system. By site-specific modification of the RNAs or the protein, the substrate- and protein-specific parameters of the RNA chaperone activity can be explored and identified.In this chapter, we present strategies on the design of labeled RNA molecules to be used to characterize the activities of an RNA-binding protein and explain how to monitor progress curves of RNA annealing and strand displacement reactions in single cuvette or well-plate scales. We provide sets of equations and models to determine and analyze different types of reactions, e.g., by calculation of first- and second-order rate constants. Likewise, we demonstrate how to exploit these simple experimental setups to elucidate elementary principles of the reaction mechanisms performed by the protein of interest by applying basic kinetic applications, such as ARRHENIUS and linear free energy relationship analyses. These approaches will be explained by providing example plots and graphs from experiments investigating the RNA chaperone activities of the RNA-binding proteins NF90-NF45 and AUF1 p45.

An RNA Thermometer Activity of the West Nile Virus Genomic 3'-Terminal Stem-Loop Element Modulates Viral Replication Efficiency during Host Switching.

The 3'-terminal stem-loop (3'SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3'SL was proposed to act as a replication silencer. The lower part of the 3'SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3'SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3'SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3'SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3'SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3'SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

Preparation and properties of recombinant rat and human procholecystokinin(57-95).

Recombinant human progastrin(6-80) binds two ferric ions with an apparent dissociation constant of 2.2 +/- 0.1 microM [Baldwin (2004) Protein J 23:65-70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin(57-95) were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin(57-95) did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin(57-95) was at least 40-fold weaker than the binding of ferric ions to progastrin(6-80).

Discovery and characterization of COVA322, a clinical-stage bispecific TNF/IL-17A inhibitor for the treatment of inflammatory diseases.

Biologic treatment options such as tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of inflammatory diseases, including rheumatoid arthritis. Recent data suggest, however, that full and long-lasting responses to TNF inhibitors are limited because of the activation of the pro-inflammatory TH17/interleukin (IL)-17 pathway in patients. Therefore, dual TNF/IL-17A inhibition is an attractive avenue to achieve superior efficacy levels in such diseases. Based on the marketed anti-TNF antibody adalimumab, we generated the bispecific TNF/IL-17A-binding FynomAb COVA322. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. COVA322 was characterized in detail and showed a remarkable ability to inhibit TNF and IL-17A in vitro and in vivo. Through its unique mode-of-action of inhibiting simultaneously TNF and the IL-17A homodimer, COVA322 represents a promising drug candidate for the treatment of inflammatory diseases. COVA322 is currently being tested in a Phase 1b/2a study in psoriasis ( ClinicalTrials.gov Identifier: NCT02243787).

Antiviral interferon-beta signaling induced by designed transcription activator-like effectors (TALE).

Here we show that designed transcription activator-like effectors (TALEs) that bind to defined areas of the interferon beta promoter are capable to induce IFN-beta expression and signaling in human cells. Importantly, TALE-mediated IFN-beta signaling occurs independently of pathogen pattern recognition but effectively prohibits viral RNA replication as demonstrated with a hepatitis C virus replicon. TALEs were thus indicated to be valuable tools in various applications addressing, for example, virus-host interactions.

A bispecific HER2-targeting FynomAb with superior antitumor activity and novel mode of action.

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets.

Contribution of extracytoplasmic function sigma factors to transition metal homeostasis in Cupriavidus metallidurans strain CH34.

Cupriavidus metallidurans strain CH34 is a highly metal-resistant bacterium that contains 11 sigma factors of the extracytoplasmic function (ECF) protein family, which can be subgrouped into the ECF:FecI 1, ECF:FecI 2, ECF:RpoE and '(ECF)' clusters. To analyze the contribution of these 11 sigma factors to metal resistance, upregulation of the respective genes was measured by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). As determined by RT-PCR, the ECF sigma factor genes were part of two- to tetra-cistronic operons, each containing genes for the sigma factor plus one or two antisigma factors. The three sigma factors RpoJ, RpoK and RpoI (ECF:FecI 1 cluster) were upregulated by Cu(II) and Ni(II), and under conditions of iron depletion. The other 8 ECF sigma factor genes were not induced by iron depletion. Strong upregulation of rpoJ and rpoK under iron depletion in a DeltarpoI mutant strain and close vicinity of rpoI to genes involved in iron siderophore metabolism marked RpoI as the primary ECF sigma factor for siderophore-mediated iron uptake. Genes for RpoO, RpoL and RpoM (ECF:FecI 2 cluster) were not upregulated by transition metal cations and influenced metal resistance only weakly. Concerning the two '(ECF)' group proteins, rpoQ was strongly upregulated by Cu(II) and deletion of rpoR led to a small decrease in copper resistance. Of the three ECF:RpoE-encoding genes, rpoP was not transcribed under the conditions tested, cnrH was upregulated by Ni(II) and essential for nickel resistance as known before. RpoE was required for full metal resistance of C. metallidurans. None of these 11 sigma factors was essential for metal resistance mediated by the cobalt, zinc and cadmium resistance determinant czc, or for its expression. However, RpoI was essential for siderophore production in C. metallidurans, and, in addition to the known role of CnrH in nickel resistance, RpoE, RpoI, RpoJ, RpoK and maybe also RpoQ are required for the outstanding transition metal resistance of this bacterium.