5S RNA Transport

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Ribosomes play a key role in the synthesis of proteins in all living cells, whether they be plant cells, animal cells, or bacteria. In eukaryotes, these complex giant macromolecules are composed of four kinds of ribosomal RNA molecules and approximately eighty different ribosomal proteins. Ribosome assembly occurs in the nucleolus of the cell, where three of the four RRNAS are synthesized. All other components of the ribosome are synthesized elsewhere and transported to the nucleolus for assembly. This research project is focused on identifying and characterizing the transport pathways and regulatory mechanisms that bring the 5S ribosomal RNA to the nucleolus for assembly into ribosomes. The research will use mutants of the cloned 5S RNA genes and their associated proteins to try to disrupt normal ribosome assembly or function in order to discern how each component might function in normal ribosome assembly. The mutated genes or their gene products (5S RNAs or 5S RNA-binding proteins) will be microinjected in Xenopus oocytes, and the gene products will be followed through the cell by way of the radioisotopic labelling and/or specific antibodies against the protein or nucleoprotein components. In vitro exchange studies will be carried out with isolated nucleoli and with isolated ribosomes, and the results will be verified in vivo through the microinjection experiments. %%% Protein synthesis is a fundamental process of all living cells. The machinery for protein synthesis is the ribosome, a complex structure consisting of many individual macromolecular components, both proteins and polyribonucleic acids (RNAs). The synthesis of ribosomes is a complex process, since the synthesis of the individual macromolecular components occurs in various parts of the cell, and sometimes at different times, yet it is necessary to bring all the different components together for assembly. The protein components of the ribosome are themselves synthesized on preexisting ribosomes, and this takes place in the cytoplasm. The ribonucleic acids are synthesized in the nucleus, since they are direct transcripts of the ribosomal genes. Three of the four RNA molecules are synthesized in a specialized region of the nucleus termed the nucleolus, first as a very long transcript which is processed into the three smaller pieces. It is to the nucleolus that all the other components are brought for assembly, including the fourth and smallest RRNA molecule, termed "5S." This 5S RNA is synthesized in the nucleus, but not in the nucleolus. Prior results from this laboratory have shown that, in Xenopus oocytes, the 5S RNA is synthesized in advance of the other RRNA components, and that after the 5S RNA is transcribed it is transported to the cytoplasm, together with its own transcription factor (a protein termed TFIIIA), and is stored there. Subsequently, it becomes dissociated from TFIIIA and associates with a different protein, L5, which is a ribosomal protein; the 5S RNA-L5 complex is then transported back to the nucleus, specifically now to the nucleolus, where it becomes assembled into the nascent ribosomes. This project will address the mechanism and regulation of this complicated nucleocytoplasmic trafficking. The work is of significance not only for understanding ribosomal biogenesis, which is itself a very important problem in biology, but also for understanding nucleocytoplasmic trafficking in general, which as a phenomenon is fundamental to much of eukaryotic cellular regulation.