Unknown,Transcriptomics,Genomics,Proteomics

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Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant: Small RNA immunoprecipitation


ABSTRACT: The Arabidopsis ARGONAUTE (AGO) protein AGO1 associates with microRNA (miRNA) and specific classes of short-interfering RNA (siRNA). AGO1-small RNA complexes recognize target RNA transcripts through base-pairing interactions and inhibit translation of target RNAs through endonucleolytic cleavage (slicing) or non-degradative mechanisms. The PIWI domain of AGO1 contains a metal-coordinating triad [Asp-Asp-His] (DDH) that is required for slicer activity. Here, we compared the activities of wild type (DDH) and slicer active-site defective (DAH) forms of AGO1 by sequencing small RNA and target transcript RNAs that co-immunoprecipitated with hemagglutinin (HA)-tagged AGO1 proteins. We found that the population of miRNA that associated with both AGO1-DDH and AGO1-DAH proteins largely overlapped, suggesting that cleavage activity does not affect miRNA maturation. In contrast, slicer-defective AGO1-miRNA complexes associated with target RNA more effectively than did wild type AGO1-miRNA. These data indicate that slicer-defective AGO proteins can be used as an approach to capture AGO-small RNA-target RNA ternary complexes more efficiently for genome-wide analyses. AGO1-DDH (wild type) AGO1-DAH (slicer mutant) proteins were immunoprecipitated (N-terminal 3xHA) from Arabidopsis (Columbia) flower (stages 1-12) lysate. Immunoprecipitations were also done from control plants transformed with vector only. Small RNA from immunoprecipitate fractions of vector, AGO1-DDH and AGO1-DAH were sequenced.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: James Carrington 

PROVIDER: E-GEOD-40258 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants.

Carbonell Alberto A   Fahlgren Noah N   Garcia-Ruiz Hernan H   Gilbert Kerrigan B KB   Montgomery Taiowa A TA   Nguyen Tammy T   Cuperus Josh T JT   Carrington James C JC  

The Plant cell 20120928 9


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 RN  ...[more]

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