Final Report Summary - SRSR (Deciphering small RNA mechanisms during sexual reproduction in Arabidopsis)
Sexual reproduction is a key step in many organisms’ life cycle that ensures the proper inheritance of traits and also allows adaptation. In both plants and metazoans, reproduction entails the complex coordination of gene expression between the different tissues involved. For example, intricate modifications of epigenetic landscapes occur during and around fertilisation ensuring zygotic totipotency, while maintaining genome integrity. RNA silencing is an ancient mechanism used by plants, animals and fungi to protect against invasive nucleic acids, including viruses and transposable elements, and to control endogenous gene expression. In recent years, key connections have been established between silencing small (s)RNA and reproduction with the discovery of piwi-associated small RNAs (piRNAs) in Drosophila controlling transposon expression. In the model plant Arabidopsis, a clear link exists between silencing sRNAs and chromatin via RNA directed DNA methylation (RdDM). However, almost nothing is known about the involvement of sRNAs in the reproductive process. To date, only a few studies have implicated sRNA in plant reproduction.
This project aims at filling this gap by studying sRNA-mediated mechanisms in tissues before and after fertilization, i.e. in the plant germline (male and female gametophyte) and in the zygote. The main goals of the SRSR projects are to:
A. Characterize the silencing machinery available during sexual reproduction
B. Establish an unbiased developmental map of reproduction-associated sRNA.
C. Study the movements of sRNA between and toward reproductive organs.
Within the two years of funding, the central question of the silencing machinery available during sexual reproduction (A) has been extensively addressed and largely solved. My colleagues and I successfully cloned and generated transgenic Arabidopsis carrying either full locus fluorescent protein fusion, full locus tagged protein and transcriptional fluorescent reporter for all Arabidopsis Argonautes (10) and Dicers (4). The analysis of the AGOs expression pattern clearly showed an asymmetry between the embryonic and the endosperm lineage (Jullien PE et al, in preparation). Most AGOs are expressed in the egg cell and embryo but not or lower in the central cell and endosperm. We have successfully established the expression map of all AGOs and DCLs during Arabidopsis reproduction. Furthermore, the tools generated will be further used to answers several other aspects of AGOs biology such as inter-cellular localization or identity of the bound sRNAs.
The movements of sRNA between and toward reproductive organs (C) has progressed, but additional efforts remain necessary. We successfully generated most of the cell-specific GFP hair-pin (GFhp) required to assess small RNA movement within reproductive organs. We observed that sRNA originating from maternal sporophytic tissue could not silenced GFP within the embryonic lineage. Expressing the GFhp in the true gametes, i.e. egg cell and sperm cell, resulted in silencing of the GFhp in a cell autonomous manner but no silencing was observed in surrounding tissue. To investigate long distance movement toward gametes we performed stem grafting using root stock expressing high level of the GFhp but no silencing of GFP was observed in male and female gametes. We concluded that sRNA either do not reach reproductive organs or to such a small quantity that they do not result in GFP silencing.
The establishment of an unbiased developmental map of reproduction-associated sRNA (B) has become a lower priority due to technical problems and increased interest in developing further the results of objective A. Indeed, during the evaluation of the silencing machinery during reproduction, we identified and characterized a new AGO of Arabidopsis called AGO3 (Jullien PE et al, to be submitted). Interestingly, AGO3 is expressed specifically at phloem termination of flowers, stamens, ovules and seeds. Down-regulation of AGO3 affects gene expression in siliques and its expression is strongly induced upon proteasome inhibition. AGO3 localized mainly to the cytoplasm, which suggests a role in post-transcriptional gene silencing (PTGS) rather than in transcriptional gene silencing (TGS). We demonstrated that AGO3 encodes a functional AGO able to bind sRNAs of 24nt in length with a 5’ nucleotide bias for adenosine. Surprisingly 24nt sRNA starting with an adenosine are known to be the one loaded by AGO4, a known TGS effector. It prompted us to investigate whether AGO3 is involved in PTGS or TGS. To do so we used a biochemical approach. We demonstrated that AGO3 fractionates with Ribosomic monosomes and polysomes, supporting a role in translational elongation. Furthermore, using mass-spectrometry of AGO3 immuno-precipitations, we found that AGO3 interacts with the translation elongation factor LOS1 but does not interact with known members of the RdDM pathway. Our results suggest that AGO3 acts to regulate gene expression by a novel RNA silencing pathway involving 24nt sRNA-directed PTGS.
This project aims at filling this gap by studying sRNA-mediated mechanisms in tissues before and after fertilization, i.e. in the plant germline (male and female gametophyte) and in the zygote. The main goals of the SRSR projects are to:
A. Characterize the silencing machinery available during sexual reproduction
B. Establish an unbiased developmental map of reproduction-associated sRNA.
C. Study the movements of sRNA between and toward reproductive organs.
Within the two years of funding, the central question of the silencing machinery available during sexual reproduction (A) has been extensively addressed and largely solved. My colleagues and I successfully cloned and generated transgenic Arabidopsis carrying either full locus fluorescent protein fusion, full locus tagged protein and transcriptional fluorescent reporter for all Arabidopsis Argonautes (10) and Dicers (4). The analysis of the AGOs expression pattern clearly showed an asymmetry between the embryonic and the endosperm lineage (Jullien PE et al, in preparation). Most AGOs are expressed in the egg cell and embryo but not or lower in the central cell and endosperm. We have successfully established the expression map of all AGOs and DCLs during Arabidopsis reproduction. Furthermore, the tools generated will be further used to answers several other aspects of AGOs biology such as inter-cellular localization or identity of the bound sRNAs.
The movements of sRNA between and toward reproductive organs (C) has progressed, but additional efforts remain necessary. We successfully generated most of the cell-specific GFP hair-pin (GFhp) required to assess small RNA movement within reproductive organs. We observed that sRNA originating from maternal sporophytic tissue could not silenced GFP within the embryonic lineage. Expressing the GFhp in the true gametes, i.e. egg cell and sperm cell, resulted in silencing of the GFhp in a cell autonomous manner but no silencing was observed in surrounding tissue. To investigate long distance movement toward gametes we performed stem grafting using root stock expressing high level of the GFhp but no silencing of GFP was observed in male and female gametes. We concluded that sRNA either do not reach reproductive organs or to such a small quantity that they do not result in GFP silencing.
The establishment of an unbiased developmental map of reproduction-associated sRNA (B) has become a lower priority due to technical problems and increased interest in developing further the results of objective A. Indeed, during the evaluation of the silencing machinery during reproduction, we identified and characterized a new AGO of Arabidopsis called AGO3 (Jullien PE et al, to be submitted). Interestingly, AGO3 is expressed specifically at phloem termination of flowers, stamens, ovules and seeds. Down-regulation of AGO3 affects gene expression in siliques and its expression is strongly induced upon proteasome inhibition. AGO3 localized mainly to the cytoplasm, which suggests a role in post-transcriptional gene silencing (PTGS) rather than in transcriptional gene silencing (TGS). We demonstrated that AGO3 encodes a functional AGO able to bind sRNAs of 24nt in length with a 5’ nucleotide bias for adenosine. Surprisingly 24nt sRNA starting with an adenosine are known to be the one loaded by AGO4, a known TGS effector. It prompted us to investigate whether AGO3 is involved in PTGS or TGS. To do so we used a biochemical approach. We demonstrated that AGO3 fractionates with Ribosomic monosomes and polysomes, supporting a role in translational elongation. Furthermore, using mass-spectrometry of AGO3 immuno-precipitations, we found that AGO3 interacts with the translation elongation factor LOS1 but does not interact with known members of the RdDM pathway. Our results suggest that AGO3 acts to regulate gene expression by a novel RNA silencing pathway involving 24nt sRNA-directed PTGS.