The Experts below are selected from a list of 17409 Experts worldwide ranked by ideXlab platform
Phillip D Zamore - One of the best experts on this subject based on the ideXlab platform.
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A 5′-uridine amplifies miRNA/miRNA* asymmetry in Drosophila by promoting RNA-induced Silencing Complex formation
Silence, 2011Co-Authors: Hervé Seitz, Jogender S Tushir, Phillip D ZamoreAbstract:MicroRNA (miRNA) are diverse in sequence and have a single known sequence bias: they tend to start with uridine (U). Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5′-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5′-U promotes miRNA loading while decreasing the loading of the miRNA*. We suggest that recognition of the 5′-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5′-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5′-nt.
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A 5'-uridine amplifies miRNA/miRNA* asymmetry in Drosophila by promoting RNA-induced Silencing Complex formation.
Silence, 2011Co-Authors: Hervé Seitz, Jogender S Tushir, Phillip D ZamoreAbstract:BACKGROUND: MicroRNA (miRNA) are diverse in sequence and have a single known sequence bias: they tend to start with uridine (U). RESULTS: Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5'-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5'-U promotes miRNA loading while decreasing the loading of the miRNA*. CONCLUSION: We suggest that recognition of the 5'-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5'-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5'-nt.
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the rna induced Silencing Complex is a mg2 dependent endonuclease
Current Biology, 2004Co-Authors: Dianne S Schwarz, Yukihide Tomari, Phillip D ZamoreAbstract:Abstract In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced Silencing Complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5′ cleavage products are readily detected for RNAi in vitro, only 3′ cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5′ and 3′ cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5′ and 3′ cleavage products in vitro; cleavage requires Mg 2+ , but not Ca 2+ , and the cleavage product termini suggest a role for Mg 2+ in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn 2+ , but not by Ca 2+ or Mg 2+ . We propose that during catalysis, a Mg 2+ ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified.
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risc assembly defects in the drosophila rnai mutant armitage
Cell, 2004Co-Authors: Yukihide Tomari, Dianne S Schwarz, Tingting Du, Benjamin Haley, Ryan Bennett, Heather A Cook, Birgit S Koppetsch, William E Theurkauf, Phillip D ZamoreAbstract:The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA Complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced Silencing Complex (RISC), which mediates target RNA destruction in RNAi. Our results suggest that armi is required for RISC maturation.
David C Baulcombe - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis argonaute1 is an rna slicer that selectively recruits micrornas and short interfering rnas
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: N Baumberger, David C BaulcombeAbstract:ARGONAUTE (AGO) RNA-binding proteins are involved in RNA Silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced Silencing Complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA- and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNA-Silencing pathways. Here, we show that AGO1 selectively recruits certain classes of short Silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgene-derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin Silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced Silencing Complex. The Slicer activity fractionates as a Complex of ≈150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.
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Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: N Baumberger, David C BaulcombeAbstract:ARGONAUTE (AGO) RNA-binding proteins are involved in RNA Silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced Silencing Complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA- and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNA-Silencing pathways. Here, we show that AGO1 selectively recruits certain classes of short Silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgene-derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin Silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced Silencing Complex. The Slicer activity fractionates as a Complex of approximately 150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.
Yukihide Tomari - One of the best experts on this subject based on the ideXlab platform.
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Native Gel Analysis for RISC Assembly
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Tomoko Kawamata, Yukihide TomariAbstract:Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced Silencing Complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor Complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.
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The microRNA pathway and cancer.
Cancer science, 2010Co-Authors: Pieter Bas Kwak, Shintaro Iwasaki, Yukihide TomariAbstract:MicroRNAs (miRNAs) are ∼22 nt long, non-coding RNAs that guide post-transcriptional gene Silencing of their target genes and regulate diverse biological processes including cancer. miRNAs do not act alone, but require assembly into RNA-induced Silencing Complex (RISC). In this review, we summarize how miRNAs are produced, assembled into RISC, and regulate target mRNAs, and discuss how the miRNA pathway is involved in cancer. (Cancer Sci 2010; 101: 2309–2315)
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Biochemical dissection of RISC assembly and function.
Nucleic acids symposium series (2004), 2009Co-Authors: Yukihide TomariAbstract:Small Silencing RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), regulate expression of their target genes via RNA-induced Silencing Complex (RISC). RISC assembly follows Complex, ordered pathways, and RISC function is as diverse as cleavage, translational repression, and deadenylation of the target. We have recently shown how siRNAs and miRNAs are assembled into distinct types of RISC, and how differently they function, using Drosophila as a model organism.
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the rna induced Silencing Complex is a mg2 dependent endonuclease
Current Biology, 2004Co-Authors: Dianne S Schwarz, Yukihide Tomari, Phillip D ZamoreAbstract:Abstract In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced Silencing Complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5′ cleavage products are readily detected for RNAi in vitro, only 3′ cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5′ and 3′ cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5′ and 3′ cleavage products in vitro; cleavage requires Mg 2+ , but not Ca 2+ , and the cleavage product termini suggest a role for Mg 2+ in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn 2+ , but not by Ca 2+ or Mg 2+ . We propose that during catalysis, a Mg 2+ ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified.
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risc assembly defects in the drosophila rnai mutant armitage
Cell, 2004Co-Authors: Yukihide Tomari, Dianne S Schwarz, Tingting Du, Benjamin Haley, Ryan Bennett, Heather A Cook, Birgit S Koppetsch, William E Theurkauf, Phillip D ZamoreAbstract:The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA Complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced Silencing Complex (RISC), which mediates target RNA destruction in RNAi. Our results suggest that armi is required for RISC maturation.
Erik J. Sontheimer - One of the best experts on this subject based on the ideXlab platform.
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RNAi: RISC Gets Loaded
Cell, 2005Co-Authors: Jonathan B. Preall, Erik J. SontheimerAbstract:When an siRNA or miRNA proceeds through the RNA-induced Silencing Complex assembly pathway, only one of the two ∼21-nucleotide RNA strands survives in the final, active Complex. In this issue of Cell, Matranga et al. (2005) and Rand et al. (2005) reveal the fate of the rejected passenger siRNA strand. Additionally, Gregory et al. (2005) define a heterotrimeric Complex from humans that appears to execute dsRNA loading, strand selection, and target mRNA cleavage activities.
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Molecular Requirements for RNA-induced Silencing Complex Assembly in the Drosophila RNA Interference Pathway
The Journal of biological chemistry, 2005Co-Authors: John W. Pham, Erik J. SontheimerAbstract:Next Section Abstract Complexes in the Drosophila RNA-induced Silencing Complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading Complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading Complex and show that the two Complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5′-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2′-position of the 5′-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2′-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2′-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.
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Assembly and function of RNA Silencing Complexes.
Nature reviews. Molecular cell biology, 2005Co-Authors: Erik J. SontheimerAbstract:In the RNA-interference pathway, double-stranded RNA induces sequence-specific mRNA degradation through the action of the RNA-induced Silencing Complex (RISC). Recent work has provided our first glimpses of the RISC-assembly pathway and uncovered the biochemical roles of critical RISC components. These advances have taken our mechanistic understanding of RNA interference to a new level and promise to improve our ability to exploit this biological process for use in experimental biology and medicine.
N Baumberger - One of the best experts on this subject based on the ideXlab platform.
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arabidopsis argonaute1 is an rna slicer that selectively recruits micrornas and short interfering rnas
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: N Baumberger, David C BaulcombeAbstract:ARGONAUTE (AGO) RNA-binding proteins are involved in RNA Silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced Silencing Complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA- and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNA-Silencing pathways. Here, we show that AGO1 selectively recruits certain classes of short Silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgene-derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin Silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced Silencing Complex. The Slicer activity fractionates as a Complex of ≈150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.
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Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: N Baumberger, David C BaulcombeAbstract:ARGONAUTE (AGO) RNA-binding proteins are involved in RNA Silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced Silencing Complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA- and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNA-Silencing pathways. Here, we show that AGO1 selectively recruits certain classes of short Silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgene-derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin Silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced Silencing Complex. The Slicer activity fractionates as a Complex of approximately 150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.
