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Deciphering the cell-specific role of Arabidopsis Argonaute 3 [RNA-seq]

ABSTRACT: ARGONAUTE (AGO) proteins are the major effectors of RNA silencing. In Arabidopsis, loading of 21-22-nt long small RNAs into AGOs results in post-transcriptional gene silencing (PTGS) by mRNA cleavage and/or translational repression. On the other hand, loading of 24-nt sRNAs results in transcriptional gene silencing (TGS) by RNA directed DNA methylation (RdDM). The Arabidopsis genome encodes 10 AGOs, which are known, for most, to belong to either pathway. Here we characterized the cell specific role of Arabidopsis AGO3. It is specifically expressed in tissue close to vascular termination and strongly accumulates in chalazal seed integuments. AGO3 encodes a functional AGO able to bind sRNAs with a preference for 24nt in length with a 5’ nucleotide bias for Adenosine. Down-regulation of AGO3 affects gene expression in siliques and its expression is strongly induced in the plant vasculature upon proteasome inhibition. Our results suggest that AGO3 might act to regulate gene expression by a novel RNA silencing pathway involving 24nt sRNA-directed PTGS.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE74721 | GEO | 2021/08/30

REPOSITORIES: GEO

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Project description:Next-generation Illumina sequencing technology was used to analyze small RNA associated with post-transcriptional gene silencing induced by intron-spliced hairpin RNA (ihpRNA) in Arabidopsis. The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine the specificity of the inhibition of gene expression. Despite its broad utility as a research tool, IR-PTGS is only a partially understood mechanism of RNA silencing in plants. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (CHS) gene fragments. The levels of PTGS were dependent on the orientation and position of the fragment in the IR construct. To investigate these differences, we characterized the sRNA profiles by Illumina sequencing of seven libraries generated from transgenic families showing different levels of IR-PTGS. Mapping of sRNA sequences to their corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation. Analyses of these patterns and peaks revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. Our observations provide new guidelines for designing constructs to increase the efficiency of IR-PTGS. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. Sequencing of small RNA from Arabidopsis plants transformed with ihpRNA constructs. Seven small RNA libraries were sequenced: Lib 0, made from non-transgenic Arabidopsis plants, and Libs 1-6, made from plants transformed with different configurations of ihpRNA.

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Deciphering the cell-specific role of Arabidopsis Argonaute 3 [RNA-seq]

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