TRBP and eIF6 homologue in Marsupenaeus japonicus play crucial roles in antiviral response

Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC). Trans-activation response RNA-binding protein (TRBP), consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP) was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6). In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi) pathway of shrimp. The double-stranded RNA binding domains (dsRBDs) B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP) were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA) and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV). These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp.     

Oligomerization activity of a double-stranded RNA-binding domain

Xenopus laevis RNA-binding protein A (Xlrbpa) is a highly conserved, ubiquitously expressed hnRNP- and ribosome-associated RNA-binding protein that contains three double stranded RNA-binding domains (dsRBDs) in tandem arrangement. A two-hybrid screen with Xlrbpa as a bait recovered Xlrbpa itself as the strongest interaction partner, indicating multimerization of this protein. To search for regions responsible for the observed interaction, we conducted two-hybrid assays with Xlrbpa deletion constructs and identified the third dsRBD of Xlrbpa as the exclusive interacting domain. Additionally, these results were confirmed by coimmunoprecipitation experiments with truncated proteins expressed both in yeast and Xenopus oocytes. In PACT, the human homologue of Xlrbpa, we could demonstrate that the third dsRBD displays the same multimerization activity. Interestingly, this domain is essential for the activation of the dsRNA-activated protein kinase PKR. Addition of RNAses to coimmunoprecipitation experiments did not affect the dimerization, suggesting that the interaction is independent of RNA-binding. We report here a homomultimerization activity of a type B dsRBD and suggest possible implications that include a model for PKR activation by PACT.     

Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen.

TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.     

RNA aptamers binding the double-stranded RNA-binding domain

Specific RNA recognition of proteins containing the double-strand RNA-binding domain (dsRBD) is essential for several biological pathways such as ADAR-mediated adenosine deamination, localization of RNAs by Staufen, or RNA cleavage by RNAse III. Structural analysis has demonstrated the lack of base-specific interactions of dsRBDs with either a perfect RNA duplex or an RNA hairpin. We therefore asked whether in vitro selections performed in parallel with individual dsRBDs could yield RNAs that are specifically recognized by the dsRBD on which they were selected . To this end, SELEX experiments were performed using either the second dsRBD of the RNA-editing enzyme ADAR1 or the second dsRBD of Xlrbpa, a homolog of TRBP that is involved in RISC formation. Several RNA families with high binding capacities for dsRBDs were isolated from either SELEX experiment, but no discrimination of these RNAs by different dsRBDs could be detected. The selected RNAs are highly structured, and binding regions map to two neighboring stem-loops that presumably form stacked helices and are interrupted by mismatches and bulges. Despite the lack of selective binding of SELEX RNAs to individual dsRBDS, selected RNAs can efficiently interfere with RNA editing in vivo.     

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.     

Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein

The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α-β-β-β-α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue.     

Expression of TAR RNA-binding protein in baculovirus and co-immunoprecipitation with insect cell protein kinase

TAR RNA-binding protein TRBP was originally isolated by its binding affinity for radiolabeled HIV-1 leader RNA. Subsequent studies have suggested that this protein is one member of a family of double-stranded RNA-binding proteins. Recent findings indicate that TRBP might function to antagonize the translational inhibitory effect that can be mediated through cellular protein kinase, PKR. Here, we report on the over-expression of a cDNA coding for TRBP in eukaryotic SF9 cells using baculovirus. We characterized the nuclear localization of TRBP in insect cells, and we demonstrate that TRBP co-immunoprecipitates with a protein in these cells antigenically related to human PKR.     

Backbone resonance assignments of the micro-RNA precursor binding region of human TRBP

TAR-RNA binding protein (TRBP) is a multidomain human protein involved in micro-RNA (miRNA) biogenesis. TRBP is a component of both the Dicer complex, which processes precursor miRNAs, and the RNA-induced silencing complex-loading complex. In addition, TRBP is implicated in the human immunodeficiency virus replication cycle and interferon-protein kinase R activity. TRBP contains 3 double-stranded RNA binding domains the first two of which have been shown to interact with miRNA precursors. Here we present the backbone resonance assignments and secondary structure of residues 19–228 of human TRBP2.     

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.     

TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress

The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTΔ13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTΔ13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTΔ13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2^−/− murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.     

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.     

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.     

Ribosome-associated factor Y adopts a fold resembling a double-stranded RNA binding domain scaffold.

Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a betaalphabetabetabetaalpha topology and represents a compact two-layered sandwich of two nearly parallel alpha helices packed against the same side of a four-stranded beta sheet. The 23 C-terminal residues of the protein are disordered. Long-range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C-terminal region of helix 1 and the loop linking this helix with strand beta2 show significant conformational exchange in the ms- micro s time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both alpha helices, which most probably constitutes an RNA binding site. The observed betaalphabetabetabetaalpha topology of pY resembles the alphabetabetabetaalpha topology of double-stranded RNA-binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs.     

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.