Arabidopsis Agonaute Ribonucleoproteins in RNA silencing

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Argonaute proteins have been implicated in RNA directed gene silencing in plants, fungi, animals and protozoans. In at least some instances they are part of the silencing effector complexes that are targeted by base pairing of an associated 21-28nt siRNA or miRNA. From analyses of AGO proteins in various organisms it seems that there may be several types of AGO-containing complex: an RNA induced specificity complex (RISC) is targeted to RNA and mediates RNA degradation and an RNA induces transcriptional silencing complex is targeted at chromosomal RNA or DNA. However, from the diversity of AGO proteins encoded in gene families it seems that there may be more than these two types of effector complex and/or that there is functional diversification of RISC and RITS complexes. In Arabidopsis, for example there are ten AGO proteins of which two, AGO1 and AGO4, act in RNA directed gene silencing. The proposed programme of work will investigate AGO protein function in silencing using Arabidopsis AGO1 as a model in the first part of the work. AGO1 is implicated in silencing by both siRNA and miRNA. In preliminary work, procedures have been developed for addition of an epitope (FLAG) tag to the amino terminus so that AGO1 can be immunprecipitated together with its associated RNAs. In an extension of this approach, other affinity tags will be added so that procedures can be optimised for purification of AGO1 together with any associated proteins and RNAs. The associated molecules will then be identified and characterised using mass spectrometry and cDNA cloning, as appropriate. The tagged proteins will also be used in immunolocalisation studies in wild type and mutant plants. It is envisaged that these studies will be informative about the identity of silencing targets and the mechanisms by which silencing is specifically targeted at RNAs. In the second part of the proposal the approaches developed for AGO1 will be applied to other Arabidopsis AGO proteins in order to find out about the diversity of silencing mechanisms. The choice of AGO proteins to be investigated in the second part of the programme will depend on an expression level that is high enough for biochemical analysis, on the functionality of the affinity tagged protein and on whether the protein is associated with siRNA or miRNA. The analysis of silencing is still at a relatively early stage and fundamental aspects of the mechanisms are still being discovered that apply in animals, plants and fungi. It is likely therefore that the findings from this work will have relevance to the practical applications of silencing in plants and animals on topics that vary from disease resistance in plants and animals, through to cancer and agriculture. It is possible for example that it will be possible to develop improved disease resistance strategies by use of transgenes to target viral genomes or to influence the expression of negative regulators of endogenous signalling pathways. It may also be possible to identify genes affecting the production of siRNa or miRNA that can be used in transgenic or conventional breeding approaches to modify growth and development.