Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissue

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. For mRNA sequencing, 2 biological samples per genotype were assessed. Samples were: ago3-3 homozygous, ago3-3 heterozygous, and Columbia 0 wilt-type samples. For sRNA sequencing, no replication was done. Three samples were analyzed: Total sRNA extracted from Arabidopsis siliques (TOTAL_smRNA), sRNA fraction which was collected from immunoprecipitated AGO3:FLAG protein (IP_AGO3), and sRNA from a control Immunoprecipitation experiment, conducted in wild-type siliques using a FLAG antibody.