Distinguishable In Vitro Binding Mode of Monomeric TRBP and Dimeric PACT with siRNA

Takahashi, Tomoko; Miyakawa, Takuya; Zenno, Shuhei; Nishi, K.; Tanokura, Masaru; Ui-Tei, Kumiko

doi:10.1371/journal.pone.0063434

Cited by 25 publications

(46 citation statements)

References 33 publications

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“…Consistent with a previous experiment (51), we could find no appreciable difference between them, although TRBP binding was low (lane 5 and lane 6 in Figure 6D and Supplementary Figure S6D). …”

Section: Resultssupporting

confidence: 92%

“…As shown previously (51), in the case of nonmodified siRNA binding, the TRBP concentration giving 50% binding of siRNA was estimated at 4.0∼5.3 × 10 − 3 μM, whereas that for siRNA with a dsDNA seed duplex (TCBA-RNA), 15∼26 μM (Figure 6C and Supplementary Figure S6C). Note that the latter is ∼500 times as high as the former concentration.…”

Section: Resultssupporting

confidence: 79%

“…Our previous experiments showed interactions between recombinant TRBP and authentic siRNA to be carried out in a two-step fashion (51). The first-step complex consists of TRBP monomer and one molecule of authentic siRNA, whereas the second-step complex, TRBP homodimer and siRNA monomer.…”

Section: Resultsmentioning

confidence: 99%

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Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells

Takahashi

Zenno

Ishibashi

et al. 2014

Nucleic Acids Research

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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.

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Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 79%

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Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells

Takahashi

Zenno

Ishibashi

et al. 2014

Nucleic Acids Research

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“…51 In contrast, the protein has been shown to directly bind small interfering (si)RNAs. [52][53][54] By electrophoresis mobility shift assay (EMSA), using a radiolabelled control siRNA and recombinant MBP or MBP-TRBP, we observed several low mobility bands, which indicates that TRBP binds to the siRNA either as a monomer or as a multimer ( Fig. 2A, left).…”

Section: Rre Binds To Trbpmentioning

confidence: 99%

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

et al. 2015

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Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and RevResponse Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

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“…In contrast, the affinity between PACT and siRNA is lower than that of the homodimerization or that between TRBP and siRNA. Thus, siRNA may be more readily incorporated into a RISC loading complex, interacting with TRBP (instead of PACT) in vivo (Takahashi et al, 2013). The cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activators (SRA) binding nuclear receptor (NR) co-regulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression (Redfern et al, 2013).…”

Section: Pact/rax and Rna Silencing Pathwaymentioning

confidence: 99%

dsRNA binding protein PACT/RAX in gene silencing, development and diseases

2014

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PACT (Protein kinase, interferon-inducible double stranded RNA dependent activator) and its murine ortholog RAX (PKR-associated protein X) were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase (PKR). Endogenous PACT/RAX activates PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. PACT/RAX heterodimerized with PKR and activated it with its third motif in the absence of dsRNA. The activation of PKR leads to enhanced eIF2α phosphorylation followed by apoptosis or inhibition of growth. Besides the role of activating PKR, PACT is associated with a ~500 kDa complex that contains Dicer, hAgo2, and TRBP (TAR RNA binding protein) and it associates with Dicer to facilitate the production of small interfering RNA. PACT/RAX plays an important role in diverse physiological and pathological processes. Pact−/− mice exhibit notable developmental abnormalities including microtia, with craniofacial ear, and hearing defects. Pact−/− mice had smaller body sizes and fertility defects, both of which were caused by defective pituitary functions. It was found that dRAX disrupted fly embryos homozygous, displayed highly abnormal commissural axon structure of the central nervous system, and 70% of the flies homozygous for the mutant allele died prior to adulthood. Using high density SNP genotyping arrays, it was found that a mutation in PRKRA (the PACT/RAX gene) is the causative genetic mutation in DYT16, a novel autosomal recessive dystonia-parkinsonism syndrome in Brazilian patients.

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Distinguishable In Vitro Binding Mode of Monomeric TRBP and Dimeric PACT with siRNA

Cited by 25 publications

References 33 publications

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

dsRNA binding protein PACT/RAX in gene silencing, development and diseases

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