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Movement of Arabidopsis thaliana inverted repeat small RNA between leaves vascular and epidermis tissues

ABSTRACT: We aimed at characterizing the identity of the silencing signal i.e. siRNA or longer dsRNA precursor thereof. We used transgenic dsRNA, endogenous dsRNA or virus infection as sources of siRNAs combined to sophisticated immunoprecipitation-based procedures coupled to deep-sequencing. We found that siRNAs populations are highly similar between incipient and recipient cells in the three systems. Additionally, a significant depletion of 5’U and 5’A content in the recipient cells suggested Argonaute-loading dependent depletion of these siRNAs during their movement over multiple cell layers. Using the PAZ-domain mutants ago1-18 and ago1-42 confirmed that the observed depletion of mobile siRNAs is the result of RISC loading. The results advocate movement of individual siRNA duplexes as opposed to their precursors and that loading into Argonaut proteins renders those small RNAs cell-autonomous.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE112929 | GEO | 2020/04/06

REPOSITORIES: GEO

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small RNA sequences from grafted Arabidopsis thaliana expressing SucSUL inverted repeat

Project description:We aimed at characterizing the identity of the silencing signal i.e. siRNA or longer dsRNA precursor thereof. We used transgenic dsRNA, endogenous dsRNA or virus infection as sources of siRNAs combined to sophisticated immunoprecipitation-based procedures coupled to deep-sequencing. We found that siRNAs populations are highly similar between incipient and recipient cells in the three systems. Additionally, a significant depletion of 5’U and 5’A content in the recipient cells suggested Argonaute-loading dependent depletion of these siRNAs during their movement over multiple cell layers. Using the PAZ-domain mutants ago1-18 and ago1-42 confirmed that the observed depletion of mobile siRNAs is the result of RISC loading. The results advocate movement of individual siRNA duplexes as opposed to their precursors and that loading into Argonaut proteins renders those small RNAs cell-autonomous.

2020-04-06 | GSE112885 | GEO

Arabidopsis thaliana small RNA from epidermis and vasculature tissues of Col-0 and ago1 mutants leaves

Project description:We aimed at characterizing the identity of the silencing signal i.e. siRNA or longer dsRNA precursor thereof. We used transgenic dsRNA, endogenous dsRNA or virus infection as sources of siRNAs combined to sophisticated immunoprecipitation-based procedures coupled to deep-sequencing. We found that although they are diluted as they move away from their sites of production, siRNAs population are highly similar between incipient and recipient cells in the three systems. Additionally, a significant depletion of 5’U and 5’A content in the recipient cells suggested Argonaute-loading dependent depletion of these siRNAs during their movement over multiple cell layers. Using the PAZ-domain mutants ago1-18 and ago1-42 confirmed that the observed depletion of mobile siRNAs is the result of RISC loading. The results advocate movement of individual siRNA duplexes as opposed to their precursors and that loading into Argonaut proteins renders those small RNAs cell-autonomous.

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Project description:We aimed at characterizing the identity of the silencing signal i.e. siRNA or longer dsRNA precursor thereof. We used transgenic dsRNA, endogenous dsRNA or virus infection as sources of siRNAs combined to sophisticated immunoprecipitation-based procedures coupled to deep-sequencing. We found that although they are diluted as they move away from their sites of production, siRNAs population are highly similar between incipient and recipient cells in the three systems. Additionally, a significant depletion of 5’U and 5’A content in the recipient cells suggested Argonaute-loading dependent depletion of these siRNAs during their movement over multiple cell layers. Using the PAZ-domain mutants ago1-18 and ago1-42 confirmed that the observed depletion of mobile siRNAs is the result of RISC loading. The results advocate movement of individual siRNA duplexes as opposed to their precursors and that loading into Argonaut proteins renders those small RNAs cell-autonomous.

2020-04-06 | GSE113029 | GEO

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics.

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Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

2012-07-12 | E-GEOD-39146 | biostudies-arrayexpress

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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

Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress [miRNA-seq]

2024-01-30 | GSE239836 | GEO

Arabidopsis AGO1 N-terminal Poly-Q domain promotes phase separation and association with stress granules during heat stress [nanoPARE]

2024-01-30 | GSE239834 | GEO

FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis [RNA-seq]

Project description:Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, AGO1 also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5’-3’ RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5’-3’ exonuclease genes XRN2 and XRN3, we found that a large number of 21-nt risiRNAs were generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlated with pre-rRNA processing defects in these mutants. We also show that these risiRNAs were loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.

2019-10-08 | GSE133411 | GEO

FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis [miRNA-seq]

2019-10-08 | GSE126482 | GEO

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