Maturation of Eukaryotic Ribosomes

The ribosome is the cellular organelle responsible for the correct translation of mRNA into the amino acid sequence of proteins and is essential for all living cells. The 80S ribosome of eukaryotes is composed of one 40S and one 60S subunit which contain different ribosomal RNA (rRNA) species and ribosomal proteins. Most of the rRNAs are transcribed in form of a single common precursor molecule which is subject a complicated series of processing and modification steps to form mature rRNAs. These processing and modification steps are coordinated by snoRNAs and protein factors which together with rRNA precursors form pre-ribosomal particles. Previous work from several groups resulted in the isolation of a number of pre-ribosomal particles consisting of different pre- rRNAs and non-ribosomal proteins that comprise intermediates of the large or small ribosomal subunit biosynthesis pathways. Most of the non-ribosomal proteins are released from the pre-ribosomal particle within the nucleolus or the nucleoplasm. However, some of them accompany the pre-ribosomal particle into the cytoplasm, where they have to be released and recycled into the nucleus to ensure an ongoing formation of ribosomal subunits. Our preliminary results indicate that the AAA-ATPase Drg1p is essential for the release of such shuttling proteins. AAA-proteins are regarded as specific chaperones that catalyze the ATP-dependent disassembly of macromolecular complexes. We found that Drg1p binds to a pre-ribosomal particle comprising the first known intermediate of the pre-60S subunit in the cytoplasm. In this project, we will characterize this intermediate in detail and will compare its composition to those of late nuclear forms of pre-ribosomal particles. This comparison will show us the alterations the pre-ribosomal particle has to go through during export from the nucleus and will allow us to identify all pre-ribosome maturation factors that accompany the pre-ribosomal particle into the cytoplasm. Using mutants lacking functional Drg1p, we will be able to determine the exact role Drg1p plays in the release of shuttling proteins. Finally our experiments will allow us to reconstruct the release of these shuttling proteins in vitro, which will enable us to investigate early cytoplasmic steps of pre-60S subunit in more detail. This project will thus be an important contribution to the understanding of the assembly pathway of this essential cellular organelle.

Ribosomes consist of a large and small subunit and are composed of ribosomal RNA and ribosomal proteins and perform the translation of mRNA into the amino acid sequence of proteins. While general aspects of the function of the ribosome in translation are quite well understood since many years, an understanding of how ribosomes are assembled emerged only in recent years and had to await refinement of affinity purification and analytical methods. Although most of the rRNAs for both subunits are initially present on a common pre-rRNA, the precursors for the large and small ribosomal subunit undergo maturation in separate pathways and are exported independently into the cytoplasm. Until several years ago, it was thought that the formation of the ribosome would be mostly finished within the nucleus and only minor adjustments would take place in the cytoplasm before joining of the subunits. However, in the last years it became obvious from work of several groups, including ours, that crucial processes for the formation of functional ribosomes only occur in the cytoplasm. This project addressed these late steps of pre-ribosome formation that take place immediately after the export of the large ribosomal subunit precursor from the nucleus into the cytoplasm. We could show that the AAA-protein Drg1, which contains two ATPase domains and forms hexamers in solution, is essential for the release of shuttling proteins. Inactivation of Drg1 by incubation of a thermo-sensitive drg1-ts strain at the restrictive temperature or depletion of the protein resulted in accumulation of all currently known shuttling proteins in the cytoplasm. Further investigations showed that these shuttling proteins failed to be released from the precursor particle which in turn resulted in a block in all further maturation steps. These results show that the cytoplasmic maturation steps of the pre-60S particle are initiated by the activity of Drg1. In line with the function of Drg1 soon after export of the pre-60S particle, we found that Drg1 interacts with components of the nuclear pore complex in vivo and in vitro. This interaction seems to be of physiological relevance since certain Drg1 alleles exhibit strong genetic interaction with genes coding for components of the nuclear pore complex and export factors. Our results therefore suggest that Drg1 binds to cytoplasmic components of the nuclear pore complex and is handed over to the pre-ribosomal particle immediately after its export to initiate the final maturation steps.