Wilhelm bernhard workshop on the cell nucleus

ISOLATION AND CHARACTERIZATION OF TOPOGENIC PROTEIN COMPLEXES PRESENT IN THE NUCLEOLUS

Hanisch C. and Schmidt-Zachmann M. S.

German Cancer Research Center, Division of Cell Biology, Heidelberg, Germany
The nucleolus which represents an accumulation of rDNA and its transcription products together with a characteristic set of proteins, is a complex nuclear substructure in which key steps of ribosome biogenesis take place. More recently, it turned out that the nucleolus is also involved in several other cellular processes such as cell cycle control, aging and mRNA export. Over the years we have reported on the identification of several constitutive nucleolar proteins (e.g. proteins NO29, NO38, NOH61), which have different roles in the complex process of ribosome biogenesis. One major aim of our recent studies will be the identification of other nucleolar proteins interacting with these components, e.g. i) by co-immuno-precipitation using the appropriate protein-specific antibodies, ii) using the tandem affinity purification (TAP) method, a technique that combines two high-affinity steps under native conditions and iii) by the yeast two-hybrid system, which in particular allows the identification of transient interaction partners. Our lab has reported the discovery and molecular characterization of the very acidic nucleolar protein, termed NO29. This protein is sequence-related to the histone-binding protein nucleoplasmin and to the major nucleolar protein NO38, i.e. it represents an additional member of the "nucleoplasmin family" (Zirwes et al. 1997, Proc. Natl. Acad. Sci. USA 94, 11387-92). Recently, we have generated a panel of mono- as well as polyclonal antibodies specifically reacting with protein NO29, which we used for immunoprecipitation experiments. Upon SDS-PAGE, a number of co-precipitating polypeptides became visible, which will be analyzed by MALDI-TOF mass spectrometry. Finally, we started to elucidate the functional roles of these non-ribosomal nucleolar proteins and their importance in maintaining the specific structures by RNA interference (RNAi).