Project description: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.
Project description: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.
Project description:AGO3 predominantly bound 24-nt sRNAs with 5â terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4 and over 60% of AGO3-associated 24-nt sRNA enriched loci were identical to those of AGO4. In addition, expression of AGO3 driven by AGO4 native promoter partially complemented AGO4 function and rescued DNA methylation defect in ago4-1 background. Examination of DNA methylation using bisulfite conversion of unmethylated cytosines in three genetic backgrounds of Arabidopsis thaliana.
Project description:AGO3 predominantly bound 24-nt sRNAs with 5â?? terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4 and over 60% of AGO3-associated 24-nt sRNA enriched loci were identical to those of AGO4. In addition, expression of AGO3 driven by AGO4 native promoter partially complemented AGO4 function and rescued DNA methylation defect in ago4-1 background. Examination of AGO3 and AGO2 bound small RNAs with/without salt stress.
Project description:AGO3 predominantly bound 24-nt sRNAs with 5’ terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4 and over 60% of AGO3-associated 24-nt sRNA enriched loci were identical to those of AGO4. In addition, expression of AGO3 driven by AGO4 native promoter partially complemented AGO4 function and rescued DNA methylation defect in ago4-1 background.
Project description:AGO3 predominantly bound 24-nt sRNAs with 5’ terminal adenine. The spectrum of AGO3-associated sRNAs was different from those bound to AGO2. By contrast, approximately 30% of AGO3-bound 24-nt sRNAs overlapped with those bound to AGO4 and over 60% of AGO3-associated 24-nt sRNA enriched loci were identical to those of AGO4. In addition, expression of AGO3 driven by AGO4 native promoter partially complemented AGO4 function and rescued DNA methylation defect in ago4-1 background.
Project description:Arabidopsis encodes ten ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22nt small RNAs (sRNA) to mediate post-transcriptional-gene-silencing (PTGS) or 24nt sRNAs to promote RNA-directed-DNA-methylation. Using full-locus constructs, we characterized the expression, biochemical properties, and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression differs drastically, with AGO3 prevailing in aerial vascular terminations and specifically in chalazal seed integuments; accordingly, AGO3 down-regulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5’-adenosine bias, but unlike most Arabidopsis AGOs - AGO2 included - it binds efficiently both 24nt and 21nt sRNAs. AGO3 immuno precipitation experiments in siliques revealed that these sRNAs mostly associate to genes and intergenic regions and not to canonical AGO targets, such as transposable elements. AGO3 localizes to the cytoplasm and co-fractionates with polysomes to possibly mediate PTGS via translation inhibition.
Project description:Parallel RNA silencing pathways regulate gene expression in plants, either by transcriptional gene silencing via RNA-dependent DNA methylation (RdDM), or by post-transcriptional silencing targeting mRNAs. Both pathways rely on distinct Dicer-like proteins to cleave double-stranded RNA into small-interfering RNAs. Experiments to determine the subcellular localization of Dicer-like proteins in Arabidopsis revealed that DCL4 is predominantly expressed as a transcriptional start site isoform that encodes a cytoplasmic protein. A second, longer DCL4 transcript isoform encodes a nuclear-localization signal and its expression is repressed by DNA methylation. Consequently this isoform is induced when promoter methylation decreases due to infection with a bacterial pathogen or during silique development. Nuclear DCL4 produces unique populations of small RNAs, called DCL4NLS isoform-dependent siRNAs (disiRNAs), which function via a post-transcriptional silencing effector, but whose precursors are generated by the RdDM pathway. Arabidopsis cells can thus respond to genome methylation changes by modulating DCL4 localization, which in turn recruits PTGS factors to reinforce RNA silencing.