Periodic Reporting for period 1 - AGOras (ARGONAUTE proteins transport routes and subcelular activities)
Periodo di rendicontazione: 2021-09-01 al 2023-08-31
Small RNAs are essential during development, stress responses, to preserve genome integrity and for general immunity to viruses. In humans, over 60% of the genes are regulated by small RNA. Deregulation of small RNA pathways are associated with a large number of human diseases, particularly cancer. To fulfill their biological function, all small RNA, are loaded into ARGONAUTE (AGO) proteins, which recognize and negatively regulate the target genes across kingdoms being themselves conserved throughout evolution. Eukaryotic AGO proteins are evolutionarily conserved but depending on the organism and the sRNA pathway involved, AGO proteins have nuclear and/or cytoplasmic localizations. Cytoplasmic AGO-sRNA complexes silence genes post-transcriptionally (PTGS) through RNA target cleavage, translational inhibition, and/or mRNA decay. In the nucleus, AGO proteins act on transcriptional gene silencing (TGS), chromatin modifications, splicing and DNA repair. While these AGO mediated processes are reasonably well understood, the transport routes involved and their regulation, are largely unknown. The overall goal of my project AGOras was to study how this AGO proteins movement could affect small RNAs and gene regulation. To achieve this, I address the following objectives:
1- To identify the signals responsible for AGO proteins subcellular localization
2- To understand how these signals are read by other proteins that move them between subcellular compartments
3- To dissect cytoplasmic and nuclear functions of AGO-sRNA pathways in plants
The interest in RNA molecules action goes beyond mere mechanistic knowledge, understanding how small RNA act is of paramount importance since RNA based vaccines or drugs are commercialized on a daily base. The outcome of AGOras will contribute to the generation of small RNA based tools to control gene expression with potential impact in therapeutic, veterinary, and agronomic sectors.
1- To identify the signals responsible for AGO proteins subcellular localization
2- To understand how these signals are read by other proteins that move them between subcellular compartments
3- To dissect cytoplasmic and nuclear functions of AGO-sRNA pathways in plants
The interest in RNA molecules action goes beyond mere mechanistic knowledge, understanding how small RNA act is of paramount importance since RNA based vaccines or drugs are commercialized on a daily base. The outcome of AGOras will contribute to the generation of small RNA based tools to control gene expression with potential impact in therapeutic, veterinary, and agronomic sectors.
To identify the signals responsible for AGO proteins subcellular localization I successfully developed an innovative computational tool that minimizes both false positives and negative results. Using the unprecedented information generated by this tool, I was able to stablish the nuclear localization and nuclear export signals (NLS and NES) of the AGO proteins in Arabidopsis.
Next, I generated Arabidopsis transgenic lines expressing the wild-type (wt) and mutant version of AGO proteins where the putative NLS/NES were mutated. Both AGOwt and AGO-NLSm/AGO-NESm where fused to GFP and expressed under their own promoter. Using confocal microscopy, I was able to stablish a map of AGO proteins functional NLS and NES in vivo by contrasting the subcellular localization of the wt and NLS/NES mutant version. Moreover, treatment of AGO proteins with commercial drugs that inhibit nuclear import or export was a complementary approach to validate our previous findings by incubating AGOwt/NLSm/NESm and studying the changes in their subcellular with confocal microscopy.
Finally, we focus on Arabidopsis AGO1 protein. Because we had thoroughly characterized the pathways for nuclear import and cytoplasmic export, we were able to generate three versions of this protein, a cytoplasmic-only, a nuclear-only and the wild-type, that it able to move between subcellular compartments. Then we isolate them and using next generation RNA sequencing we identify the different small RNA that are loaded in each of the subcellular compartments, thus describing the complexes that can initiate small RNA-target regulation. This allows us to develop a model framework to understand how AGO proteins control the small RNA cellular partitioning and their final mode of action in plants.
The work and results of this project were widely disseminated. I have presented them among colleagues during institutional seminaries, program retreats, and for the scientific community in specific and general congresses in Spain, Germany, Austria, Argentina and Japan.
Next, I generated Arabidopsis transgenic lines expressing the wild-type (wt) and mutant version of AGO proteins where the putative NLS/NES were mutated. Both AGOwt and AGO-NLSm/AGO-NESm where fused to GFP and expressed under their own promoter. Using confocal microscopy, I was able to stablish a map of AGO proteins functional NLS and NES in vivo by contrasting the subcellular localization of the wt and NLS/NES mutant version. Moreover, treatment of AGO proteins with commercial drugs that inhibit nuclear import or export was a complementary approach to validate our previous findings by incubating AGOwt/NLSm/NESm and studying the changes in their subcellular with confocal microscopy.
Finally, we focus on Arabidopsis AGO1 protein. Because we had thoroughly characterized the pathways for nuclear import and cytoplasmic export, we were able to generate three versions of this protein, a cytoplasmic-only, a nuclear-only and the wild-type, that it able to move between subcellular compartments. Then we isolate them and using next generation RNA sequencing we identify the different small RNA that are loaded in each of the subcellular compartments, thus describing the complexes that can initiate small RNA-target regulation. This allows us to develop a model framework to understand how AGO proteins control the small RNA cellular partitioning and their final mode of action in plants.
The work and results of this project were widely disseminated. I have presented them among colleagues during institutional seminaries, program retreats, and for the scientific community in specific and general congresses in Spain, Germany, Austria, Argentina and Japan.
The results obtained in this project, contributed to move the field forward in several directions. I developed an original tool to predict subcellular localization signals and have used it to build a comprehensive and experimentally validated map of Arabidopsis AGO proteins subcellular localization and movements. Moreover, this methodological approach has the potential to be applied to other AGO proteins, including humans, thus it could be used to boost our understanding of AGO shuttling mechanism across kingdoms. Furthermore, the algorithm underlying the tool can easily be used to improve predictions of post-translational modifications which could also affect subcellular localization or protein stability.
At the same time, the understanding of AGO proteins nuclear import and export routes, have allowed me to design mutant AGO proteins confined to specific subcellular compartment. Combining this tool with next generation sequencing techniques, has provided me with detail picture of nuclear, cytoplasmic, and shuttling AGO-small RNA complexes. This data, which delivered a global view of small RNA intracellular movement and functions, can be used to improve crops.
The outcome of AGOras can contribute to the Societal Challenge 2 of the Work Programme H2020: Food Security, Sustainable Agriculture and Forestry, Marine, Maritime and Inland Water Research and the Bioeconomy since it addresses key challenges that our planet is facing for the years to come, i.e. ensuring food security for the population. Crops can be infected by virus resulting in necrosis, withering and death, thus producing large economic losses. AGO proteins can provide viral defense and viruses can be found either in the nucleus or the cytoplasm of plants cells. Indeed, AGOras has set a collection of AGO proteins with nuclear or cytoplasmic only destination, that could help protect plants from a variety of viral infections. Since AGO proteins are deeply conserved in plants, it could be envisaged that the results derived from AGOras could be easily implemented on the development of virus resistant crops in the near future.
At the same time, the understanding of AGO proteins nuclear import and export routes, have allowed me to design mutant AGO proteins confined to specific subcellular compartment. Combining this tool with next generation sequencing techniques, has provided me with detail picture of nuclear, cytoplasmic, and shuttling AGO-small RNA complexes. This data, which delivered a global view of small RNA intracellular movement and functions, can be used to improve crops.
The outcome of AGOras can contribute to the Societal Challenge 2 of the Work Programme H2020: Food Security, Sustainable Agriculture and Forestry, Marine, Maritime and Inland Water Research and the Bioeconomy since it addresses key challenges that our planet is facing for the years to come, i.e. ensuring food security for the population. Crops can be infected by virus resulting in necrosis, withering and death, thus producing large economic losses. AGO proteins can provide viral defense and viruses can be found either in the nucleus or the cytoplasm of plants cells. Indeed, AGOras has set a collection of AGO proteins with nuclear or cytoplasmic only destination, that could help protect plants from a variety of viral infections. Since AGO proteins are deeply conserved in plants, it could be envisaged that the results derived from AGOras could be easily implemented on the development of virus resistant crops in the near future.