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| Status |
Public on Sep 28, 2012 |
| Title |
Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant: Small RNA immunoprecipitation |
| Organism |
Arabidopsis thaliana |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
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.
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| Contributor(s) |
Carbonell A, Fahlgren N, Garcia-Ruiz H, Gilbert KB, Montgomery TA, Nguyen T, Cuperus JT, Carrington JC |
| Citation(s) |
23023169 |
| Submission date |
Aug 21, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
James C Carrington |
| E-mail(s) |
jcarrington@danforthcenter.org
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| Phone |
314-587-1202
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| Organization name |
Donald Danforth Plant Science Center
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| Lab |
James C. Carrington
|
| Street address |
975 North Warson Road
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63132 |
| Country |
USA |
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| Platforms (1) |
| GPL13222 |
Illumina HiSeq 2000 (Arabidopsis thaliana) |
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| Samples (3) |
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| This SubSeries is part of SuperSeries: |
| GSE40259 |
Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant |
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| Relations |
| SRA |
SRP014967 |
| BioProject |
PRJNA173789 |
