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.
Highlights
In small RNA-directed RNA silencing pathways, ARGONAUTE (AGO) proteins associate with small RNA to target and silence transcripts (Kim et al, 2009)
The third position of the catalytic triad was mutated to an Asp in AGO1 and AGO7 and to a His in AGO2
As AGO2 is involved in antiviral silencing, this will be discussed separately from AGO1, AGO7, and AGO10, which associate with miRNAs that affect developmental processes
Summary
In small RNA-directed RNA silencing pathways, ARGONAUTE (AGO) proteins associate with small RNA to target and silence transcripts (Kim et al, 2009). Interactions between AGO–small RNA complexes and target RNA usually lead to repression of the transcripts through either direct cleavage (slicing) or through other mechanisms, such as target destabilization or translational repression (recently reviewed in Huntzinger and Izaurralde, 2011). (1) the identification of endogenous factors, such as SUO, which is required for miRNA-directed translational repression (Yang et al, 2012); (2) the association of AGO1 with ribosomes in an mRNA-dependent manner (Lanet et al, 2009); and (3) the possibility that miRNA targets may be subject to deadenylation and decapping in plants (Brodersen et al, 2008) has led to the idea that translation repression could be a widespread regulatory mechanism in plants (Brodersen et al, 2008)
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