Slicer activity of Arabidopsis AGO1 in siRNA biogenesis and miRNA target decay
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2000
ORGANISM(S): Arabidopsis thaliana
SUBMITTER: Laura Arribas-Hernandez
PROVIDER: E-MTAB-4529 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. Nuclear AGO1 is loaded with miRNAs and exported to the cytosol where it associates to the rough ER to conduct miRNA-mediated translational repression, mRNA cleavage and biogenesis of phased siRNAs. These latter, as well as other cytosolic siRNAs, are loaded into cytosolic AGO1, but in which compartment this happens is not known. Moreover, the effect of stress on AGO1 localization is still unclear. Here, we show that heat stress (HS) promotes AGO1 protein accumulation, which co-localizes with components of the siRNA bodies and of stress granules (SGs). AGO1 does not need SGS3, a key component of siRNA bodies, to efficiently form condensates during HS. Instead, we found that the still poorly characterized N-terminal Poly-Q domain of AGO1, which contains a prion-like domain, is sufficient to undergo phase separation. Moreover, an exposure of 1 hour to HS only moderately affected AGO1 loading by miRNAs and target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting its activity in reprograming gene expression during stress. Collectively, our work shed new light on the impact of high temperatures on the main effector of RNA silencing in plants.
2024-01-30 | GSE239834 | GEO
Project description:In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. Nuclear AGO1 is loaded with miRNAs and exported to the cytosol where it associates to the rough ER to conduct miRNA-mediated translational repression, mRNA cleavage and biogenesis of phased siRNAs. These latter, as well as other cytosolic siRNAs, are loaded into cytosolic AGO1, but in which compartment this happens is not known. Moreover, the effect of stress on AGO1 localization is still unclear. Here, we show that heat stress (HS) promotes AGO1 protein accumulation, which co-localizes with components of the siRNA bodies and of stress granules (SGs). AGO1 does not need SGS3, a key component of siRNA bodies, to efficiently form condensates during HS. Instead, we found that the still poorly characterized N-terminal Poly-Q domain of AGO1, which contains a prion-like domain, is sufficient to undergo phase separation. Moreover, an exposure of 1 hour to HS only moderately affected AGO1 loading by miRNAs and target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting its activity in reprograming gene expression during stress. Collectively, our work shed new light on the impact of high temperatures on the main effector of RNA silencing in plants.
2024-01-30 | GSE239836 | GEO
Project description:In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. Nuclear AGO1 is loaded with miRNAs and exported to the cytosol where it associates to the rough ER to conduct miRNA-mediated translational repression, mRNA cleavage and biogenesis of phased siRNAs. These latter, as well as other cytosolic siRNAs, are loaded into cytosolic AGO1, but in which compartment this happens is not known. Moreover, the effect of stress on AGO1 localization is still unclear. Here, we show that heat stress (HS) promotes AGO1 protein accumulation, which co-localizes with components of the siRNA bodies and of stress granules (SGs). AGO1 does not need SGS3, a key component of siRNA bodies, to efficiently form condensates during HS. Instead, we found that the still poorly characterized N-terminal Poly-Q domain of AGO1, which contains a prion-like domain, is sufficient to undergo phase separation. Moreover, an exposure of 1 hour to HS only moderately affected AGO1 loading by miRNAs and target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting its activity in reprograming gene expression during stress. Collectively, our work shed new light on the impact of high temperatures on the main effector of RNA silencing in plants.
2024-01-30 | GSE239833 | GEO
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. 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.
2012-09-28 | E-GEOD-40258 | biostudies-arrayexpress
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.
2012-09-28 | GSE40256 | GEO
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.
2012-09-28 | GSE40258 | GEO
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
Project description:To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by IlluminaM-CM-"M-BM-^@M-BM-^Ys technology. We identified some additional miRNAs, collateral miRNAs encoded in known miRNA precursors, phased smRNA clusters and nat-siRNAs. Organ specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. We used extracts from Arabidopsis flowers, leaves and roots as well as ten-day old seedlings to purify smRNAs associated with AGO1 and AGO4 protein complexes using a two-step immunoprecipitation method.
2011-04-19 | E-GEOD-28591 | biostudies-arrayexpress
Project description:microRNAs (miRNAs) are a class of small silencing RNAs that have regulatory roles in gene expression. miRNAs interact with Argonaute (AGO) proteins to form effector complexes that can cleave target mRNAs or repress their translation. Rice encodes four AGO1 homologs (AGO1a, AGO1b, AGO1c, and AGO1d). We used an RNAi approach to knock down the four AGO1s. The RNAi lines displayed pleiotropic developmental phenotypes and had increased accumulation of miRNA targets, suggesting the involvement of AGO1s in the miRNA pathway. Three of the AGO1s (AGO1a, AGO1b, and AGO1c complexes) were purified and further characterized. We showed that the three AGO1s all have a strong preference for binding small RNAs (sRNAs) with 5’ U and have Slicer activity. We catalogued the sRNAs in each AGO1 complex by deep sequencing, and found all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s in the function of these miRNAs. Intriguingly, we also found a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting that there is also functional specialization among the rice AGO1s. Four samples, total extract and three AGO1 complexes were analyzed
2009-11-23 | E-GEOD-18250 | biostudies-arrayexpress
Project description:To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illuminaâs technology. We identified some additional miRNAs, collateral miRNAs encoded in known miRNA precursors, phased smRNA clusters and nat-siRNAs. Organ specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs.
2011-04-19 | GSE28591 | GEO