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Multiple Argonaute complexes orchestrate epigenetic silencing in the Arabidopsis germline [RNA-Seq]

ABSTRACT: We report AGO4/5/6/9-bound sRNAs map to pol iv-dependent sRNA clusters in the Arabidopsis thaliana male sexual lineage that regulates gene expression in meiocytes. We found that AGO4/6/9 but not AGO5 participate in RdDM in meiocytes, and AGO5 plays a subtle role in repressing TEs silenced by AGO4/6/9 and Pol IV in meiocytes.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE342115 | GEO | 2026/07/31

REPOSITORIES: GEO

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Multiple Argonaute complexes orchestrate epigenetic silencing in the Arabidopsis germline [Bisulfite-Seq]

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DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV

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RNA-directed DNA methylation involves co-transcriptional small RNA-guided slicing of Pol V transcripts in Arabidopsis.

Project description:Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide (nt) small RNAs (siRNAs) that bind ARGONAUTE4 (AGO4) and target genomic regions for silencing. It also requires non-coding RNAs transcribed by RNA POLYMERASE V (Pol V), that likely serve as scaffolds for binding of AGO4/siRNA complexes. Here we utilized a modified global nuclear run-on (GRO) protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5’ adenine found in many AGO4-bound 24-nt siRNAs and was eliminated in an siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expressing wild-type AGO4 in ago4/6/9 was able to restore slicing of Pol V transcripts but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required the little understood elongation factor SPT5L. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

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