Structural insights into RNA processing by the human RISC-loading complex

Wang, Hongwei; Noland, Cameron L.; Siridechadilok, Bunpote; Taylor, David W.; Ma, Enbo; Felderer, Karin; Doudna, Jennifer A.; Nogales, Eva

doi:10.1038/nsmb.1673

Cited by 228 publications

(229 citation statements)

References 34 publications

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“…Our results are in contrast to the TAR-mediated disruption of the TRBP-Dicer interaction observed in the context of overexpressed TRBP, Dicer and TAR. 33 In our assays, interactions between the key endogenous proteins of the RISC, TRBP, Dicer and Ago2 3,5,69,70 were maintained despite high expression levels of either TAR or RRE RNAs (Fig. 3A).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…The PAZ and N-terminal domain have been shown to undergo extensive structural rearrangements when progressing from the binary to the ternary complex. 12,21,22,31 In contrast, the MID-PIWI lobe of Ago is conformational stable and the 5′ end of the guide strand and the guide/target hybrid bound via the MID-PIWI domains can be clearly assigned in the crystal structure. the most pronounced FRET shift indicating that the distance between nucleotide 18 and this position changed dramatically.…”

Section: Conformational Changes On Progressing From the Binary To Thementioning

confidence: 99%

Single-molecule FRET supports the two-state model of Argonaute action

et al. 2013

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“…growing number of different assemblies including small nuclear ribonucleoproteins (18,37) and spliceosomes (3,38), a box C/D small ribonucleoprotein (39), RNA editing complexes (13), survival of motor neuron complexes (40), the pre-mRNA 3Ј processing complex (41), the RNA-induced silencing complex (RISC)-loading complex (42), the RNA polymerase (10), the DNA gyrase (10), the DNA topoisomerase (10), a multifunctional replication protein (43), basal transcription factors (44), and p53 (45).…”

Section: Discussionmentioning

confidence: 99%

Merging Molecular Electron Microscopy and Mass Spectrometry by Carbon Film-assisted Endoproteinase Digestion

Richter

Sander

Golas

et al. 2010

Molecular & Cellular Proteomics

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Many fundamental processes in the cell are performed by complex macromolecular assemblies that comprise a large number of proteins. Numerous macromolecular assemblies are structurally rather fragile and may suffer during purification, resulting in the partial dissociation of the complexes. These limitations can be overcome by chemical fixation of the assemblies, and recently introduced protocols such as gradient fixation during ultracentrifugation (GraFix) offer advantages for the analysis of fragile macromolecular assemblies. The irreversible fixation, however, is thought to render macromolecular samples useless for studying their protein composition. We therefore developed a novel approach that possesses the advantages of fixation for structure determination by single particle electron microscopy while still allowing a correlative compositional analysis by mass spectrometry. In this method, which we call "electron microscopy carbon film-assisted digestion", macromolecular assemblies are chemically fixed and then adsorbed onto electron microscopical carbon films. Parallel, identically prepared specimens are then subjected to structural investigation by electron microscopy and proteomics analysis by mass spectrometry of the digested sample. As identical sample preparation protocols are used for electron microscopy and mass spectrometry, the results of both methods can directly be correlated. In addition, we demonstrate improved sensitivity and reproducibility of electron microscopy carbon film-assisted digestion as compared with standard protocols. We show that sample amounts of as low as 50 fmol are sufficient to obtain a comprehensive protein composition of two model complexes. We suggest our approach to be an optimization technique for the compositional analysis of macromolecules by mass spectrometry in general. Molecular & Cellular Proteomics 9:1729 -1741, 2010.An increasing number of proteins are nowadays recognized to fulfill their cellular tasks as part of macromolecular machines (1). Recent advances in the isolation procedures allow purification of many of these assemblies for further biochemical and structural analysis (2-4). Thereby, MS of peptides derived by digestion of the proteins of the assemblies (5, 6) or MS of intact assemblies (7, 8) is used to determine the protein composition, whereas electron microscopy (EM) 1 can be used to provide information about the structure of the assembly using computational averaging and reconstruction techniques (9). Available protocols to combine information derived by MS and EM therefore either analyze the endoproteolytic digestion products (10 -13) or the intact assembly (14). However, the sample preparation protocols typically differ between EM and MS. Although for MS the sample is either directly digested in solution (6), subsequent to gel electrophoretic separation of the sample (15-17) or used in an intact form (7,8), EM typically requires the adsorption of the sample to carbon film (13, 18 -20). In the case of sample heterogeneity, the utilization of thes...

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Structural insights into RNA processing by the human RISC-loading complex

Cited by 228 publications

References 34 publications

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

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

Single-molecule FRET supports the two-state model of Argonaute action

Merging Molecular Electron Microscopy and Mass Spectrometry by Carbon Film-assisted Endoproteinase Digestion

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