title: Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants.
abstract: In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.
authors.affiliation: Donald Danforth Plant Science Center, St. Louis, MO 63132, USA.
authors.forename: Alberto
authors.initials: A
authors.lastname: Carbonell
authors.forename: Noah
authors.initials: N
authors.lastname: Fahlgren
authors.forename: Hernan
authors.initials: H
authors.lastname: Garcia-Ruiz
authors.forename: Kerrigan B
authors.initials: KB
authors.lastname: Gilbert
authors.forename: Taiowa A
authors.initials: TA
authors.lastname: Montgomery
authors.forename: Tammy
authors.initials: T
authors.lastname: Nguyen
authors.forename: Josh T
authors.initials: JT
authors.lastname: Cuperus
authors.forename: James C
authors.initials: JC
authors.lastname: Carrington
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D055106:Genome-Wide Association Study; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D010641:Phenotype; D010935:Plant Diseases / Q000821:virology; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D017800:Potyvirus / Q000502:physiology; D055550:Protein Stability; D034622:RNA Interference; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D019076:Transgenes
chemical_list: C117457:AGO1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D018749:RNA, Plant; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C486145:ZIPPY protein, Arabidopsis; C559855:argonaute 2 protein, Arabidopsis