The unique dual targeting of AGO1 by two types of PRMT enzymes promotes phasiRNA loading in Arabidopsis thaliana

Arginine / R methylation (R-met) of proteins is a widespread post-translational modification (PTM), deposited by a family of protein arginine / R methyl transferase enzymes (PRMT). Regulations by R-met are involved in key biological processes deeply studied in metazoan. Among those, post-transcriptional gene silencing (PTGS) can be regulated by R-met in animals and in plants. It mainly contributes to safeguard processes as protection of genome integrity in germ lines through the regulation of piRNA pathway in metazoan, or response to bacterial infection through the control of AGO2 in plants. So far, only PRMT5 has been identified as the AGO / PIWI R-met writer in higher eukaryotes. We uncovered that AGO1, the main PTGS effector regulating plant development, contains unique R-met features among the AGO / PIWI superfamily, and outstanding in eukaryotes. Indeed, AGO1 contains both symmetric (sDMA) and asymmetric (aDMA) R-dimethylations and is dually targeted by PRMT5 and by another type I PRMT in Arabidopsis thaliana . We showed also that loss of sDMA didn't compromise AtAGO1 subcellular trafficking in planta. Interestingly, we underscored that AtPRMT5 specifically promotes the loading of a set of phasiRNA in AtAGO1. All our observations bring to consider this dual regulation of AtAGO1 in plant development and response to environment, and pinpoint the complexity of AGO1 post-translational regulation. Overall design: To investigate the impact of atprmt5 mutations on total sRNA accumulation and AtAGO1 sRNA loading in reproductive tissues. To do so, we carried out anti-AGO1 immunoprecipitations performed on close buds of A. thaliana, using Col0, atprmt5-2 and atprmt5-6 mutants. A fraction of inputs (total extracts) and all the IP products are used for sRNA extraction. The experiment includes biological duplicates.