Wilhelm bernhard workshop on the cell nucleus

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.