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| Status |
Public on Sep 28, 2012 |
| Title |
Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant: Transcript immunoprecipitation |
| Organism |
Arabidopsis thaliana |
| Experiment type |
Expression 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. Immunoprecipitate fractions were treated with nuclease P1 before cleanup to trim free RNA ends, and therefore enrich samples in AGO1 target sites. All samples were treated with Ribominus (Life Technologies) to reduce ribosomal RNA abundance. Transcript fragments from two replicates of AGO1-DDH and AGO1-DAH immunoprecipitates were sequenced. Ribominus-treated total RNA (input controls) was also sequenced for each replicate.
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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
|
| Phone |
314-587-1202
|
| Organization name |
Donald Danforth Plant Science Center
|
| Lab |
James C. Carrington
|
| Street address |
975 North Warson Road
|
| 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 (8)
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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 |
SRP014970 |
| BioProject |
PRJNA173788 |
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