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

RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Protein Science (Impact Factor: 2.85). 01/2011; 20(1):118-30. DOI: 10.1002/pro.543
Source: PubMed

ABSTRACT 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.

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Available from: Takashi Nagata, Jul 18, 2014
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    • "Human TRBP contains two dsRNA-binding domains, dsRBD1 and dsRBD2, both of which include α-β-β-β-α–folds and three amino acids presumably involved in dsRNA binding in any ∼16-nt region (60,61). Mutagenesis along with structural analysis indicated that each TRBP domain is capable of interacting with dsRNA via at least two sites in minor and major grooves, which are <11 nt apart from each other and close to the distance between the distal ends of siRNA subdomains A and C (see the upper margin in Figure 3). "
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    ABSTRACT: Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5' proximal nucleotides, which include seed sequence (positions 2-8 from the 5' end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19-21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15-18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.
    Nucleic Acids Research 02/2014; 42(8). DOI:10.1093/nar/gku153 · 9.11 Impact Factor
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    • "As mentioned earlier, both TRBP and PACT interact with Dicer using their C-terminal dsRBD3 and bind to dsRNA substrates with the two N-terminal dsRBD1-2 (5,16,17,23). To investigate the mechanism of dsRBPs’ effects on Dicer, we designed two chimeric proteins, T12P3 and P12T3, in which the third domain of TRBP and PACT has been swapped (Figure 4A). "
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    ABSTRACT: During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer-TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer-dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.
    Nucleic Acids Research 05/2013; 41(13). DOI:10.1093/nar/gkt361 · 9.11 Impact Factor
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    • "The second dsRBD of human TRBP contains a KR-helix motif that can strongly bind to dsRNA, whereas N-terminal dsRBD minimally binds RNA and directs guide strand selection from microRNA duplexes in humans [35], [39]. Yamashita et al. found that the structure of dsRBD1 and dsRBD2 of human TRBP were similar, and could bind to siRNA, with dissociation constants of 220 and 113 nM respectively [40]. A lysine- and arginine-rich motif termed TR13 (KKLAKRNAAAKMLLR) derived from the dsRBD2 of human TRBP is necessary to bind HIV trans-activation response(TAR) RNA upper-stem/loop site, and the two Arg and Lys residues are important for the RNA-binding activity [35], [36], [37]. "
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    ABSTRACT: 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.
    PLoS ONE 01/2012; 7(1):e30057. DOI:10.1371/journal.pone.0030057 · 3.23 Impact Factor
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