Wilhelm bernhard workshop on the cell nucleus

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.