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 slicerindependent 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, slicerdefective 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.

mRNA Decay of Most Arabidopsis miRNA Targets Requires Slicer Activity of AGO1

“…Expression of slicer-deficient AGO1 enhances the morphological and molecular phenotypes of the hypomorphic ago1-25 allele (Carbonell et al, 2012). These dominantnegative effects were probably due to competition of the slicer-deficient protein with the residual endogenous AGO1 activity of ago1-25, by sequestration of miRNAs or miRNA-target complexes (Carbonell et al, 2012). A dominant-negative effect on wild-type plants was also described.…”

“…Although a low level of slicer activity remained detectable in the E803A mutant (Arribas-Hernández et al, 2016), its phenotype was identical to that of mutants in the other three catalytic residues in all aspects that distinguish them from the null allele. Expression of slicer-deficient AGO1 enhances the morphological and molecular phenotypes of the hypomorphic ago1-25 allele (Carbonell et al, 2012). These dominantnegative effects were probably due to competition of the slicer-deficient protein with the residual endogenous AGO1 activity of ago1-25, by sequestration of miRNAs or miRNA-target complexes (Carbonell et al, 2012).…”

Plant MicroRNAs Display Differential 3' Truncation and Tailing Modifications That Are ARGONAUTE1 Dependent and Conserved Across Species

“…Consistent with this, AGO1 and HESO1 colocalize (G. Ren, M. Xie, C. Vinovskis, and B. Yu, unpublished data). We also examined immunoprecipitated miRNAs from an AGO1 slicer-defective mutant (Carbonell et al, 2012) and observed little impact on miR158 tailing, indicating that AGO1 slicer activity is not required to modulate 39 modifications (see Supplemental Figure 6B online). Such 39 modifications occurring without target cleavage are reminiscent of D. melanogaster miRNA truncation and tailing that occurs when extensively matched by a synthetic target mRNA (Ameres et al, 2010).…”

“…Data for Columbia, heso1-1, hen1-8, and heso1-1 hen1-8 libraries are from Zhao et al (2012b). IP data from the wild type and catalytic mutants of AGO1 are from Carbonell et al (2012).…”

Identification and characterization of a novel geminivirus with a monopartite genome infecting apple trees

“…Indeed, both viruses and viroids are targeted by the plant RNA-silencing machinery that, through the activity of Dicer-like proteins (DCLs), generates virus-and viroid-derived sRNAs (v-sRNA and vd-sRNA, respectively) of 21-24 nt, which are loaded into AGO (argonaute) proteins to guide the sequence-specific degradation or methylation of cognate RNAs and DNA, respectively (Carbonell et al, 2012;Mallory & Vaucheret, 2010;Minoia et al, 2014;Miozzi et al, 2013;Navarro et al, 2012). Both v-sRNAs and vd-sRNAs accumulating in infecting tissues are therefore molecular markers of ongoing infections by viral and subviral agents.…”