Wilhelm bernhard workshop on the cell nucleus

Proteomic and functional analysIs of the Arabidopsis nucleolus

Shaw P¹, Beven A¹, Clark G², Lam Y³, Andersen J⁴, Mann M⁴, Lamond A³ , Brown J<sup>2

1</sup>John Innes Centre, Norwich, UK; ²Scottish Crop Research Institute, Dundee, Scotland, UK; ³University of Dundee, Dundee, Scotland, UK; ⁴University of Southern Denmark, Odense, Denmark
The nucleolus is traditionally recognised as the site of rDNA transcription, rRNA processing and ribosomal subunit assembly, but clearly is involved in a range of other activities and processes. For example, the nucleolus is involved in aspects of processing or export of some mRNAs, the signal recognition particle (SRP) and some tRNAs, and the processing and assembly of telomerase RNP and U6snRNAs. The nucleolus is often closely associated with Cajal bodies which function in the maturation and recycling of snRNPs; assembly, processing or trafficking to the nucleolus of snoRNPs; 3' end processing of some histone mRNAs, and modification of snRNAs. The proteome of the human nucleolus continues to be investigated both in terms of its composition and the function of the identified proteins. We have carried out a proteomic analysis of Arabidopsis nucleoli purified from protoplasts, allowing a comparative proteomic analysis of two widely separated higher eukaryotes to identify common and species-specific nucleolar components, and to better assess commonality of the unknown proteins found in both analyses. We have so far identified around 200 different proteins, including ribosomal proteins, known nucleolar and RNA-binding proteins, putative DNA/chromatin binding proteins, histone acetylases/deacetylases, DEAD box helicases, splicing and translation factors, putative RNA transport factors, snoRNP core proteins and many hitherto unknown proteins. We are transferring available cDNAs for the identified proteins in a high throughput way into Gateway vectors in order to express GFP-protein fusions in Arabidopsis cells and verify the nucleolar location of the proteins. To date, the high percentage of GFP fusions showing nucleolar labelling highlights the quality of the nucleolar preparation. Detailed analysis of their sub-nucleolar location should also provide clues about potential functions of the proteins and the different processes in which they are involved.