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Global identification of Arabidopsis thaliana ARGONAUTE1-associated small RNA.

ABSTRACT: Small RNA from total RNA (input fraction) and immunoprecipitated HA-AGO1-complexes (IP fraction) were identified using high-throughput sequencing-by-synthesis. Two replicate input fraction samples were used as controls for two replicate IP fraction samples. Total RNA from input fractions and small RNA that co-immunoprecipitated with HA-AGO1 complexes were size fractionated by polyacrylamide gel electrophoresis to recover 18-24 nucleotide small RNA. 3' and then 5' adaptors were serially ligated on to each small RNA followed by PAGE purification. RT-PCR was used to convert RNA to DNA amplicons. Amplicons were sequenced using the Illumina GAIIx platform. Resulting reads were parsed and mapped to the A. thaliana genome (TAIR9) using the CASHX pipeline.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: James Carrington

PROVIDER: E-GEOD-22252 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis.

Cuperus Josh T JT Carbonell Alberto A Fahlgren Noah N Garcia-Ruiz Hernan H Burke Russell T RT Takeda Atsushi A Sullivan Christopher M CM Gilbert Sunny D SD Montgomery Taiowa A TA Carrington James C JC

Nature structural & molecular biology 20100618 8

RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors cont ...[more]

PMID: 20562854

Similar Datasets

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2015-04-06 | E-GEOD-64911 | biostudies-arrayexpress

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Global identification of Arabidopsis thaliana ARGONAUTE1-associated small RNA.

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The Arabidopsis F-box protein FBW2 degrades AGO1 to avoid spurious loading of illegitimate small RNA

Project description:RNA silencing is a conserved mechanism in eukaryotes and is involved in development, heterochromatin maintenance and defense against viruses. In plants, ARGONAUTE1 (AGO1) protein plays a central role in both microRNA (miRNA) and small interfering RNA (siRNA)-directed silencing and its expression is regulated at multiple levels. Here, we report that the F-box protein FBW2 targets proteolysis of AGO1 by a CDC48-mediated mechanism. We found that FBW2 assembles an SCF complex that recognizes the MID-PIWI domain of AGO1 and requires its C-terminal domain containing a GW motif for AGO1 turnover. We showed that FBW2 has a preference for the unloaded and for some mutated forms of AGO1 protein. While FBW2 loss of function does not lead to strong growth or developmental defects, it significantly increases RNA silencing activity. Interestingly, under conditions in which small RNA production or accumulation is affected, the failure to degrade AGO1 in fbw2 mutants becomes more deleterious for the plant. Hence, we showed that the non-degradable AGO1 protein assembles high molecular complexes and binds illegitimate small RNA leading to the cleavage of new target genes that belong to stress responses and cellular metabolic processes. Therefore, the control of AGO1 homeostasis by ubiquitin ligases, plays an important quality control to avoid off-target cleavage.

2022-04-01 | PXD024840 | Pride

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

Project description: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. 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.

2012-09-28 | E-GEOD-40256 | biostudies-arrayexpress

Profiling of Argonaute (AGO) 2-associated sRNAs after infection of bacterial pathogen Pseudomonas syringae

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2011-05-10 | GSE26161 | GEO

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