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.

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.

Characterization of Soft Amyloid Cores in Human Prion-Like Proteins.

Prion-like behaviour is attracting much attention due to the growing evidences that amyloid-like self-assembly may reach beyond neurodegeneration and be a conserved functional mechanism. The best characterized functional prions correspond to a subset of yeast proteins involved in translation or transcription. Their conformational promiscuity is encoded in Prion Forming Domains (PFDs), usually long and intrinsically disordered protein segments of low complexity. The compositional bias of these regions seems to be important for the transition between soluble and amyloid-like states. We have proposed that the presence of cryptic soft amyloid cores embedded in yeast PFDs can also be important for their assembly and demonstrated their existence and self-propagating abilities. Here, we used an orthogonal approach in the search of human domains that share yeast PFDs compositional bias and exhibit a predicted nucleating core, identifying 535 prion-like candidates. We selected seven proteins involved in transcriptional or translational regulation and associated to disease to characterize the properties of their amyloid cores. All of them self-assemble spontaneously into amyloid-like structures able to propagate their polymeric state. This provides support for the presence of short sequences able to trigger conformational conversion in prion-like human proteins, potentially regulating their functionality.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins. Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

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.

Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles.

The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells.

NF45 and NF90 Bind HIV-1 RNA and Modulate HIV Gene Expression.

A previous proteomic screen in our laboratory identified nuclear factor 45 (NF45) and nuclear factor 90 (NF90) as potential cellular factors involved in human immunodeficiency virus type 1 (HIV-1) replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1 which is required for Tat-dependent trans-activation of viral gene expression. In this study the roles of NF45 and NF90 in HIV replication were investigated through overexpression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 and NF90 diminished the enhancement of HIV infection and gene expression. Both proteins were found to interact with the HIV RNA. RNA decay assays demonstrated that NF90, but not NF45, increased the half-life of the HIV RNA. Overall, these studies indicate that both NF45 and NF90 potentiate HIV infection through their RNA binding domains.

Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA.

Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3' untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs.

Identification of lncRNA MEG3 Binding Protein Using MS2-Tagged RNA Affinity Purification and Mass Spectrometry.

Long noncoding RNAs (lncRNAs) are nonprotein coding transcripts longer than 200 nucleotides. Recently in mammals, thousands of long noncoding RNAs have been identified and studied as key molecular players in different biological processes with protein complexes. As a long noncoding RNA, maternally expressed gene 3 (MEG3) plays an important role in many cellular processes. However, the mechanism underlying MEG3 regulatory effects remains enigmatic. By using the specific interaction between MS2 coat protein and MS2 RNA hairpin, we developed a method (MS2-tagged RNA affinity purification and mass spectrometry (MTRAP-MS)) to identify proteins that interact with MEG3. Mass spectrometry and gene ontology (GO) analysis showed that MEG3 binding proteins possess nucleotide binding properties and take part in transport, translation, and other biological processes. In addition, interleukin enhancer binding factor 3 (ILF3) and poly(A) binding protein, cytoplasmic 3 (PABPC3) were validated for their interaction with MEG3. These findings indicate that the newly developed method can effectively enrich lncRNA binding proteins and provides a strong basis for studying MEG3 functions.

NF90 isoforms, a new family of cellular proteins involved in viral replication?

The Nuclear Factor 90 (NF90) and its isoforms constitute a family of proteins that can interact with double-stranded (ds) RNA, through its dsRNA binding motifs. Due to various potential translational events such as alternative splicing, the human Interleukin enhancer binding factor 3 (ilf3) gene codes for multifunctional proteins that are NF90 and its isoforms, involved in transcription, translation, mRNA export and microRNA biogenesis. These proteins can act as cellular partners affecting viral replication and they are also implicated in host defense. As a result of these numerous functions, these protein isoforms have been given various names over the years, leading to confusion in determining their specific functions. In this review we focus on the role of the human NF90 protein isoforms in DNA and RNA virus replication.

NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold.

Nuclear factors NF90 and NF45 form a complex involved in a variety of cellular processes and are thought to affect gene expression both at the transcriptional and translational level. In addition, this complex affects the replication of several viruses through direct interactions with viral RNA. NF90 and NF45 dimerize through their common 'DZF' domain (domain associated with zinc fingers). NF90 has additional double-stranded RNA-binding domains that likely mediate its association with target RNAs. We present the crystal structure of the NF90/NF45 dimerization complex at 1.9-Å resolution. The DZF domain shows structural similarity to the template-free nucleotidyltransferase family of RNA modifying enzymes. However, both NF90 and NF45 have lost critical catalytic residues during evolution and are therefore not functional enzymes. Residues on NF90 that make up its interface with NF45 are conserved in two related proteins, spermatid perinuclear RNA-binding protein (SPNR) and zinc-finger RNA-binding protein (Zfr). Using a co-immunoprecipitation assay and site-specific mutants, we demonstrate that NF45 is also able to recognize SPNR and Zfr through the same binding interface, revealing that NF45 is able to form a variety of cellular complexes with other DZF-domain proteins.

L-Ilf3 and L-NF90 traffic to the nucleolus granular component: alternatively-spliced exon 3 encodes a nucleolar localization motif.

Ilf3 and NF90, two proteins containing double-stranded RNA-binding domains, are generated by alternative splicing and involved in several functions. Their heterogeneity results from posttranscriptional and posttranslational modifications. Alternative splicing of exon 3, coding for a 13 aa N-terminal motif, generates for each protein a long and short isoforms. Subcellular fractionation and localization of recombinant proteins showed that this motif acts as a nucleolar localization signal. Deletion and substitution mutants identified four arginines, essential for nucleolar targeting, and three histidines to stabilize the proteins within the nucleolus. The short isoforms are never found in the nucleoli, whereas the long isoforms are present in the nucleoplasm and the nucleoli. For Ilf3, only the posttranslationally-unmodified long isoform is nucleolar, suggesting that this nucleolar targeting is abrogated by posttranslational modifications. Confocal microscopy and FRAP experiments have shown that the long Ilf3 isoform localizes to the granular component of the nucleolus, and that L-Ilf3 and L-NF90 exchange rapidly between nucleoli. The presence of this 13 aminoacid motif, combined with posttranslational modifications, is responsible for the differences in Ilf3 and NF90 isoforms subcellular localizations. The protein polymorphism of Ilf3/NF90 and the various subcellular localizations of their isoforms may partially explain the various functions previously reported for these proteins.

Production of HIV particles is regulated by altering sub-cellular localization and dynamics of Rev induced by double-strand RNA binding protein.

Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1-dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication.

Nuclear factor 90 negatively regulates influenza virus replication by interacting with viral nucleoprotein.

Interactions between host factors and the viral replication complex play important roles in host adaptation and regulation of influenza virus replication. A cellular protein, nuclear factor 90 (NF90), was copurified with H5N1 viral nucleoprotein (NP) from human cells in which NP was transiently expressed and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. In vitro coimmunoprecipitation of NF90 and NP coexpressed in HEK 293T cells or individually expressed in bacterial and HEK 293T cells, respectively, confirmed a direct interaction between NF90 and NP, independent of other subunits of the ribonucleoprotein complex. This interaction was prevented by a mutation, F412A, in the C-terminal region of the NP, indicating that the C-terminal of NP is required for NF90 binding. RNase V treatment did not prevent coprecipitation of NP and NF90, which demonstrates that the interaction is RNA binding independent. After small interfering RNA knockdown of NF90 expression in A549 and HeLa cells, viral polymerase complex activity and virus replication were significantly increased, suggesting that NF90 negatively affects viral replication. Both NP and NF90 colocalized in the nucleus of virus-infected cells during the early phase of infection, suggesting that the interaction between NF90 and NP is an early event in virus replication. Quantitative reverse transcription-PCR showed that NF90 downregulates both viral genome replication and mRNA transcription in infected cells. These results suggest that NF90 inhibits influenza virus replication during the early phase of infection through direct interaction with viral NP.

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function.

BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. RESULTS: Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. CONCLUSION: The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.