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

Takahashi, Tomoko; Zenno, Shuhei; Ishibashi, Osamu; Tajima, Toshihiro; Saigo, Kaoru; Ui-Tei, Kumiko

doi:10.1093/nar/gku153

Cited by 28 publications

(23 citation statements)

References 59 publications

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“…Therefore we examined whether KUNV sfRNA was capable of directly interacting with a dsRNA-binding RISC complex component (Takahashi et al, 2014). As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Flavivirus sfRNA suppresses antiviral RNA interference in cultured cells and mosquitoes and directly interacts with the RNAi machinery

et al. 2015

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Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA interference (RNAi) pathway, a major antiviral response in mosquitoes. In this study, we demonstrate that two flaviviruses, Dengue virus and Kunjin virus, significantly repress siRNA-mediated RNAi in infected human cells as well as during infection of the mosquito vector Culex quinquefasciatus. Arthropod-borne flaviviruses generate a small structured non-coding RNA from the viral 3’ UTR referred to as sfRNA. Analysis of infections with a mutant Kunjin virus that is unable to generate appreciable amounts of the major sfRNA species indicated that RNAi suppression was associated with the generation of the non-coding sfRNA. Co-immunoprecipitation of sfRNA with RNAi mediators Dicer and Ago2 suggest a model for RNAi suppression. Collectively, these data help to establish a clear role for sfRNA in RNAi suppression and adds to the emerging impact of viral long non-coding RNAs in modulating aspects of anti-viral immune processes.

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“…Therefore we examined whether KUNV sfRNA was capable of directly interacting with a dsRNA-binding RISC complex component (Takahashi et al, 2014). As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Flavivirus sfRNA suppresses antiviral RNA interference in cultured cells and mosquitoes and directly interacts with the RNAi machinery

et al. 2015

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“…Additional factors known to be involved in cytoplasmic RNAi include TNRC6A (GW182) (Jakymiw et al 2005;Eulalio et al 2008;Nishi et al 2013;, Dicer (Bernstein et al 2001;Ha and Kim 2014), and TAR RNA binding protein (TRBP) (Daniels and Gatignol 2012;Wilson and Doudna 2013;Takahashi et al 2014). TNRC6A influences the subcellular localization of AGO2 and is critical for miRNA-guided silencing.…”

Section: Introductionmentioning

confidence: 99%

Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

Kalantari

Hicks

et al. 2016

RNA

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Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.

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“…The RNA sequences were designed based on the fact that the TRBP2-dsRBD1 is known to interact with miR-16-1 duplex (9). The miR-16-1 duplex contains a bulge (created by an unpaired uridine), and an internal loop (created by a A•A mismatch) in the dsRNA-binding region (40), thereby creating a deviation from the A-form helical structure ( Figure 1A). The passenger strand of miR-16-1 was mutated to generate the following three RNA sequence mutants: i) miR-16-1-M with only the mismatch ( Figure 1B); ii) miR-16-1-B with only the bulge ( Figure 1C); and iii) miR-16-1-D which forms a perfect duplex by removing both the bulge and the internal loop ( Figure 1D).…”

Section: Rna Sequence Design and Sample Preparationmentioning

confidence: 99%

Role of protein dynamics in enthalpy-driven recognition of topologically distinct dsRNAs by dsRBDs

Paithankar

Chugh

2019

Preprint

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Taking leads from the fact that a handful of double-stranded RNA-binding domains (dsRBDs) interact with a massive number of topologically distinct double-stranded RNAs (dsRNAs) in crucial biological pathways, and to understand the adaptability required by dsRBDs to target the pool of dsRNA substrates, we employed two independent model dsRBDs in an ITC and NMR spectroscopy based study. Our previous study revealed the presence of microsecond timescale dynamics in RNAbinding regions in the two dsRBDs studied. Results from ITC-based titrations showed that the binding of dsRBD with topologically distinct dsRNAs is enthalpy-driven, with each dsRNA-dsRBD pair having distinct combination of enthalpy-entropy yielding a similar change in free energy. We also show that the each of the dsRNA used binds to the dsRBD in a unique mode. Further, intrinsic conformational exchange present in the RNA-binding regions of the apo-dsRBD was shown to quench upon binding with a dsRNA, while conformational exchange got induced at the residues that are in close proximity to where exchange was present in the apo-protein. This apparent relay of conformational exchange from one site to the other site upon dsRNA-binding thus suggests the importance of intrinsic dynamics to adapt to target a variety of dsRNA-shapes. Statement of SignificanceThis study reports that the interaction between dsRBDs and dsRNAs is enthalpy-driven, is perturbed by subtle changes in dsRNA shapes, and exhibits a classic case of enthalpy-entropy compensation.Further, the intrinsic microsecond timescale conformational exchange present in the apo-dsRBD was observed to get quenched upon RNA-binding. An apparent relay of conformational exchange was also observed from quenched sites to spatially proximal sites upon RNA-binding, suggesting highly adaptive nature of the dsRBD, a critical feature required to target topologically distinct dsRNA.Choudhary and Mr. Mayuresh for providing access to ITC facility at UM-DAE CEBS, Mumbai.

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

Cited by 28 publications

References 59 publications

Flavivirus sfRNA suppresses antiviral RNA interference in cultured cells and mosquitoes and directly interacts with the RNAi machinery

Flavivirus sfRNA suppresses antiviral RNA interference in cultured cells and mosquitoes and directly interacts with the RNAi machinery

Stable association of RNAi machinery is conserved between the cytoplasm and nucleus of human cells

Role of protein dynamics in enthalpy-driven recognition of topologically distinct dsRNAs by dsRBDs

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