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.

FUNCTIONAL PROTEOMIC ANALYSIS OF PLANT NUCLEOLAR PHOSPHOPROTEINS RELATED TO CELL PROLIFERATION

González-Camacho, F., and Medina, F.J.

Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
The soluble fraction of nuclear proteins is a functionally significant fraction, since it has been shown that it contains ribonucleoproteins active in the nuclear RNA metabolism. The aim of this work was to detect variations associated with cell proliferation, by comparing 2D- proteomes obtained from the soluble fraction of onion nuclei in actively proliferating root meristematic cells versus non-meristematic root cells. In particular, we have studied the physico-chemical features of the major nucleolar protein NopA100, a highly phosphorylated nucleolin-like protein. A total of 388 spots were quantified in meristematic nuclei, while only 215 were detected in non-meristematic nuclei. The comparison of both proteomes resulted in the determination of specific spots for each proliferative state and those which were common to both cases. Furthermore, among these latter, we could discriminate quantitative differences. Interestingly, relevant well-known nucleolar proteins, such as RNA polymerase I, B23 and the nucleolin-like protein NopA100, exhibited a significant increase in proliferating cells. By Western blotting with anti-NopA100 antibody, 17 spots, 100 kDa in molecular mass, were detected in the meristematic sample. All the spots detected form a cluster through a pI range of 4.3-6.6. This cluster gives account of different states of phosphorylation exhibited by the protein in dependence of the nucleolar activity and the cell cycle phases. On the contrary, only 8 spots were found in the extract from non-meristematic nuclei, whose pI range was shortened to 4.8-6.1. This indicates a substantially lower amount of phosphorylation variants, associated with the drop of the proliferation capacity and of the nucleolar activity. We have also analyzed the bidimensional AgNOR staining pattern of this fraction in order to identify in plant cells the stained protein spots, which are commonly recognized to be phosphoproteins, markers of proliferation. Other than corroborating that NopA100, the onion nucleolin-like protein, is an Ag-NOR protein, we were interested in the characterization of the plant homologue to the nucleolar phosphoprotein B23. It was revealed as two clusters of acidic spots, 43 and 42 kDa respectively in molecular mass. These features are not totally coincident with those described for mammalian cells. The use of protein fractionation procedures with functional significance and the location of candidate spots by indirect techniques are an advantageous alternative to random selection procedures for proteomic studies involving further mass spectrometry analysis.

TRAFFIC OF NEWLY FORMED RIBOSOMES IN THE NUCLEUS

¹Politz J.C., ²Tuft R.A. and ¹Pederson, T.

¹Program in Cell Dynamics, ¹Department of Biochemistry & Molecular Pharmacology, and ²Dept. of Physiology, University of Massachusetts Medical School, Worcester, USA.
An aspect of ribosome synthesis that is not well understood is the spatial pathway of intranuclear ribosome traffic. We designed oligonucleotides complementary to 28S rRNA regions that are known to lie distant from the decoding site on the assembled 60S ribosomal subunit. When taken up by living rat myoblast cells, these oligos became localized in the nucleoli (as well as in the cytoplasm as expected). An in situ reverse transcription assay revealed that the oligos were actually hybridized to their endogenous RNA targets in both nucleoli and the cytoplasm. We then coupled caged fluorescein to these oligos in order to follow the movements of newly formed 60S ribosomal particles in the nucleus of living cells, after uncaging the fluorescent groups attached to the hybridized probes. The signals moved out from the uncaged nucleoli in all directions, with no evidence of directed transport along any spatially distinct paths. When the uncaging beam was directed to nucleoplasmic regions in order to track 60S subunits that had already left the nucleoli, the signals were again observed to be moving in all directions, behaving as a mean square displacement phenomenon, i.e. consistent with a diffusive process. A most surprising observation was that after exiting a nucleolus, some of the ribosomal particles were observed to enter another nucleolus. Similarly, some of the particles that were uncaged in the nucleoplasm also displayed this return to nucleoli. These results establish that ribosome transport out of the nucleolus and within the nucleoplasm has a diffusive component and that, surprisingly, ribosomes re-visit nucleoli before nuclear export. Thus, the ability to examine ribosome movements in the nuclei of living cells has revealed that the process of a nascent ribosome getting from its birthplace, the nucleolus, to the cytoplasm is not as direct and simple a journey as might have been anticipated.
RECRUITMENT OF RIBOSOMAL PROTEINS TO PRE-RIBOSOMES AS STUDIED BY EXPRESSION OF GFP-FUSION PROTEINS IN LIVING CELLS

Krüger, T., and Scheer, U.

Department of Cell and Developmental Biology, Theodor-Boveri-Institute, University of Würzburg, Am Hubland, Würzburg, Germany
A question of fundamental importance is how the cascade of biochemical reactions involved in ribosome biosynthesis is spatially organized and integrated into nucleolar structure. Ribosome biogenesis is a vectorial process which begins in the fibrillar portion of the nucleolus and continues into the surrounding granular component (GC). After passing through the GC where the late steps of pre-rRNA processing and of ribosome assembly occur, the almost mature ribosomal subunits are eventually released into the nucleoplasm and exit the nucleus through the nuclear pore complexes. By confocal immunocytochemistry and expression of fluorescent nucleolar marker proteins we have resolved the three basic nucleolar domains which are related to rDNA transcription, early and late steps of pre-ribosome assembly (fibrillar centers, dense fibrillar component and granular component, respectively). Hence, early and late binding ribosomal proteins should reveal different localizations within the nucleolus. We have expressed several GFP-tagged ribosomal proteins and examined their intranucleolar distribution in living mammalian cells. Ribosomal protein L4 has been described as a primary binding protein which already binds to the nascent pre-rRNA transcripts (Chooi and Leiby, PNAS 78:4823, 1981). Surprisingly, L4 as well as the other ribosomal proteins examined so far (S9, L23, L5) were detectable exclusively in the GC indicating that they all associate with preribosomal particles at a relatively late stage of their maturation pathway. L10 (=QM) has been described as cytoplasmic ribosomal protein in human cells (Nguyen et al., J. Cell. Biochem. 68:281, 1998). Upon inhibition of nuclear export of ribosomal subunits by the antibiotic Leptomycin B, GFP-L10 fusion proteins as well as endogenous L10 accumulated in the nucleoplasm and the GC of nucleoli. Thus, mammalian L10 appears to have a late nuclear function compatible with its proposed role in the nuclear export of the large ribosomal subunits in yeast.