title: The Slicer Activity of ARGONAUTE1 Is Required Specifically for the Phasing, Not Production, of Trans-Acting Short Interfering RNAs in Arabidopsis.
abstract: ARGONAUTE1 (AGO1) mediates posttranscriptional silencing by microRNAs (miRNAs) and short interfering RNAS (siRNAs). AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets, but current models also highlight the roles of slicing in formation of siRNAs and siRNA-AGO1 complexes. miRNA-guided slicing is required for biogenesis of phased, trans-acting siRNAs (tasiRNAs), whose cleaved precursor fragments are converted to double-stranded RNA by RNA-dependent RNA polymerase 6 (RDR6). In addition, unwinding of duplex siRNA bound to AGO1 requires passenger strand cleavage in vitro. In this study, we analyze how mutation of four metal ion-coordinating residues of Arabidopsis thaliana AGO1 affects slicer activity in vitro and siRNA function in vivo. We show that while all four residues are required for slicer activity, they do not contribute equally to catalysis. Moreover, passenger strand cleavage is required for assembly of active AGO1-siRNA complexes in vivo, and many AGO1-bound siRNAs are trimmed in the absence of slicer activity. Remarkably, seedlings defective in AGO1 slicer activity produce abundant siRNAs from tasiRNA loci in vivo. These siRNAs depend on RDR6 and SUPPRESSOR OF GENE SILENCING3, but unlike wild-type tasiRNAs, they are unphased. These results demonstrate that slicing is solely required for phase definition of tasiRNAs, and they strongly support recruitment of RDR6 by AGO1 rather than by cleavage fragments.
authors.affiliation: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: Laura
authors.identifier: 0000-0003-0605-0407
authors.initials: L
authors.lastname: Arribas-Hernández
authors.affiliation: Swiss Federal Institute of Technology (ETH), 8092 Zurich, Switzerland.
authors.forename: Antonin
authors.initials: A
authors.lastname: Marchais
authors.affiliation: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Poulsen
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Bettina
authors.identifier: 0000-0002-9811-473X
authors.initials: B
authors.lastname: Haase
authors.affiliation: Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Judith
authors.initials: J
authors.lastname: Hauptmann
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Vladimir
authors.identifier: 0000-0002-0352-2547
authors.initials: V
authors.lastname: Benes
authors.affiliation: Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany.
authors.forename: Gunter
authors.initials: G
authors.lastname: Meister
authors.affiliation: Department of Biology, University of Copenhagen, DK-2200 Copenhagen N, Denmark pbrodersen@bio.ku.dk.
authors.forename: Peter
authors.identifier: 0000-0003-1083-1150
authors.initials: P
authors.lastname: Brodersen
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism
chemical_list: C117457:AGO1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D035683:MicroRNAs; D018749:RNA, Plant; D034741:RNA, Small Interfering; C514575:RDR6 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase