Final Report Summary - AGOIM (Characterization of the interactions and modifications affecting AtAGO1 function)
RNA silencing is a key mechanism controlling gene expression via 19-24 nucleotide (nt) small RNAs (sRNAs). In plants, sRNAs regulate important developmental and reproductive processes as well as adaptive responses to biotic and abiotic stresses. In addition, silencing mediated by sRNAs is part of the plant “immune system”, conferring protection against virus infection. ARGONAUTES (AGOs) are one of the main components of the small RNA (sRNA) silencing pathway. They are the core members of the RNA-induced silencing complex (RISC), which silence target genes at the chromatin or at the mRNA level. Among the 10 AGO paralogs of the model plant Arabidopsis thaliana (AGO1-10), AGO1 is the main effector of the micro RNA (miRNA) pathway and is also associated with various classes of small interfering RNAs (siRNAs) acting post-transcriptionally, including transgene-, virus-derived and trans-acting siRNAs (tasiRNAs). Despite its importance in plant physiology and defense, scarce information is available regarding the modulation of AGO1 activity at the protein level. Most specifically, we are interested in understand how post-translational modifications and protein-protein interactions affect AGO1 function, and consequently the RNA silencing pathway.
One post-translational modification that might affect AGO1 is arginine methylation. Arabidopsis AGO1 has ten residues, which could be methylated. In order to test the effects of protein methylation on AGO1 function we have generated a mutant version of AGO1, in which the amino acids involved were replaced (AGO1met-). This construct has being used, together with a wild type version of AGO1 (AGO1wt) to complement mutant plants and also in transient experiments using Nicotiana benthamiana plants. An agro-infiltration assay in N. benthamina suggests that lack of methylation might stabilize AGO1. The same pattern could not be observed so clearly in stable transgenic A. thaliana plants. The fact these plants carry more then one copy of AGO1 could help to explain why this stability aspect could not be noticed with the same intensity in Arabidopisis. Alternatively, low variation in AGO1 levels are expected when expressed constitutively due to the tightly regulation that this protein suffers, as consequence of its key role in development. Although we could not see any major effect of the lack of methylation in most aspects of AGO1 physiology, we could identify proteins that might be part of the RISC complex only when AGO1 is methylated. We are now analyzing the role of these proteins in the RNA silencing pathway.
Proteins interactions might also affect the way AGO1 works. One protein that might interact with AGO1 is the Tudor-containing protein TUDOR-SN (TSN). In A. thaliana, there are two TSN proteins (TSN1 and TSN2). We have investigated whether the lack of TSN1/2 has any effect on AGO1. Therefore we have analyzed tsn1/2 double mutants regarding many aspects of AGO1 biology. We could not detect any direct influence of TSN1/2 on AGO1. However, it could be that this would be only evident when certain stresses are applied. We have also tested which role TSN1/2 plays in the proper function of the viral suppressor of RNA silencing P19. Unfortunately, we could not reach any conclusion due to silencing of the P19 gene.
We were also interested to test the hypothesis that protein interactions might allow AGO1 to trigger the production of secondary sRNAs, such as tasiRNAs. We have performed several 2-O-methyl and Immune-precipitation (IP) assays followed by mass-spectrometry (MS) to identify proteins that might interact with AGO1 when this is able to start transitivity (production of secondary sRNAs). We have identified and validated one protein that might have this role. We have also identified interactors that might be constitutive members of the RISC complex. T-DNA knockout mutants from the respective genes are being analyzed in order to clarify the role of these proteins in the RNA silencing pathway.
Overall, our results aim to improve our knowledge about how AGO1 function is regulated and also how this regulation affects the function of AGO1. Being the main effector protein of RNA silencing in plants, our findings could have a positive influence in the development of new approaches to combat virus infection and also the development of new tools based on RNA silencing. We could also help in the future advance of our understanding of silencing and plant biology. At last, but not last important, we hope that with this results we will positively impact the attractiveness of Europe as a center of excellence for science.
One post-translational modification that might affect AGO1 is arginine methylation. Arabidopsis AGO1 has ten residues, which could be methylated. In order to test the effects of protein methylation on AGO1 function we have generated a mutant version of AGO1, in which the amino acids involved were replaced (AGO1met-). This construct has being used, together with a wild type version of AGO1 (AGO1wt) to complement mutant plants and also in transient experiments using Nicotiana benthamiana plants. An agro-infiltration assay in N. benthamina suggests that lack of methylation might stabilize AGO1. The same pattern could not be observed so clearly in stable transgenic A. thaliana plants. The fact these plants carry more then one copy of AGO1 could help to explain why this stability aspect could not be noticed with the same intensity in Arabidopisis. Alternatively, low variation in AGO1 levels are expected when expressed constitutively due to the tightly regulation that this protein suffers, as consequence of its key role in development. Although we could not see any major effect of the lack of methylation in most aspects of AGO1 physiology, we could identify proteins that might be part of the RISC complex only when AGO1 is methylated. We are now analyzing the role of these proteins in the RNA silencing pathway.
Proteins interactions might also affect the way AGO1 works. One protein that might interact with AGO1 is the Tudor-containing protein TUDOR-SN (TSN). In A. thaliana, there are two TSN proteins (TSN1 and TSN2). We have investigated whether the lack of TSN1/2 has any effect on AGO1. Therefore we have analyzed tsn1/2 double mutants regarding many aspects of AGO1 biology. We could not detect any direct influence of TSN1/2 on AGO1. However, it could be that this would be only evident when certain stresses are applied. We have also tested which role TSN1/2 plays in the proper function of the viral suppressor of RNA silencing P19. Unfortunately, we could not reach any conclusion due to silencing of the P19 gene.
We were also interested to test the hypothesis that protein interactions might allow AGO1 to trigger the production of secondary sRNAs, such as tasiRNAs. We have performed several 2-O-methyl and Immune-precipitation (IP) assays followed by mass-spectrometry (MS) to identify proteins that might interact with AGO1 when this is able to start transitivity (production of secondary sRNAs). We have identified and validated one protein that might have this role. We have also identified interactors that might be constitutive members of the RISC complex. T-DNA knockout mutants from the respective genes are being analyzed in order to clarify the role of these proteins in the RNA silencing pathway.
Overall, our results aim to improve our knowledge about how AGO1 function is regulated and also how this regulation affects the function of AGO1. Being the main effector protein of RNA silencing in plants, our findings could have a positive influence in the development of new approaches to combat virus infection and also the development of new tools based on RNA silencing. We could also help in the future advance of our understanding of silencing and plant biology. At last, but not last important, we hope that with this results we will positively impact the attractiveness of Europe as a center of excellence for science.