Ribosomes are ribonucleoprotein (RNP) particles that catalyze the translation of the genetic information of messenger RNA (mRNA) into proteins. The biogenesis of eukaryotic ribosomes is a highly complex process that starts in the nucleolus where the ribosomal RNA (rRNA) is synthesized by RNA Polymerases I and III. Three of the four rRNA molecules are produced as one precursor molecule (pre-rRNA) that contains spacer sequences that are not part of mature ribosomal subunits but are removed during ribosome biogenesis. Besides these processing events, rRNA is heavily modified.
In the course of ribosome assembly, the numerous (around 80) ribosomal proteins (r-proteins) and the rRNA (precursors) join together in a highly dynamic and defined manner. Initial interactions of most of the r-proteins to preribosomes occur already in the nucleolus or in the nucleus; some seem to be incorporated in the cytoplasm where the some final maturation events happen. Most of the r-proteins are required for an efficient accumulation of functional ribosomal subunits in the cytoplasm. Interestingly, depletion of essential LSU r-proteins resulted not in one common but in several different pre-rRNA processing phenotypes indicating their requirement for specific maturation events. In vitro reconstitution experiments on prokaryotic ribosomal subunits (whose structures are highly similar to the ones of their eukaryotic counterparts) helped to analyze the hierarchical interrelationships between the individual r-protein assembly events. In eukaryotic cells, the production of ribosomal subunits does not occur in a “self assembly” mechanism but requires numerous transiently interacting proteins, the so called ribosome biogenesis factors, and many small nucleolar RNAs (snoRNAs). Most of our current knowledge on eukaryotic ribosome biogenesis comes from studies in the yeast S.cerevisiae. Highly resolved structural information of mature yeast ribosomes (which is available since a few years) contributed to a better understanding of ribosome function but also ribosome biogenesis. Although the knowledge of the binding sites of the structural components (r-proteins) in the mature ribosomal subunits might not fully reflect how these “endpoints” are established in vivo, the final structure is very helpful to better understand features like hierarchical interrelationship of r-protein assembly events and/or their requirement for specific maturation events.
In this work, the assembly process of the 46 r-proteins of the yeast large ribosomal subunit was aimed to be characterized in terms of several previously fragmentary or unresolved aspects. By comparing the respective amounts of the individual LSU r-proteins in differently maturated LSU precursors to each other and/or to the ones in mature LSUs, the assembly characteristics of each of the 46 LSU r-proteins was addressed applying a combination of affinity purification (ex vivo) and quantitative mass spectrometry. In a complementary approach, the interaction of a number of essential LSU r-proteins with the nascent particles was investigated in a comparative way by affinity purification of epitope tagged LSU r-proteins and quantitative analysis of the co-purified (pre-) rRNA species. In order to better understand the requirement of individual LSU r-proteins for certain assembly or maturation events, various LSU r-protein expression mutants were then used to investigate changes in the composition of the mutant preribosomes. Both, changes in the association of other LSU r-proteins and LSU ribosome biogenesis factors were analyzed. The results were then compared to previously published data from in vivo and in vitro experiments.
The comparative analyses on the composition of differently maturated LSU precursors indicated that most LSU r-proteins seem to already start interacting with preribosomes of early maturation stages. In average, the affinity of these initial interactions seems to be rather low, though. The binding strength of most LSU r-proteins is then stabilized in the course of ribosome maturation. Therefore, the stable incorporation of most LSU r-proteins seems to be a multistep, rather than a one step event. Some LSU r-proteins however showed a specific assembly behavior which was characterized by an underrepresentation in early (or early and intermediately) maturated LSU precursors. Their (stable) incorporation seems to occur at later stages; for one group of LSU r-proteins in the cytoplasm.
Clear evidences for hierarchical interrelationships among LSU r-protein assembly events in vivo could be deduced from the analyses of the RPL mutants. They can be categorized into two kinds of effects. One effect observed after depletion of most LSU r-proteins was characterized by a partial destabilization of the mutant preribosomes in the direct neighborhood of the depleted LSU r-protein or in the same secondary structure domain. Second, more general effects which can be related to the depletion phenotype of the respective r-protein were observed in most cases. A comparison to the results of previously published in vitro reconstitution experiments of prokaryotic LSUs revealed clear differences between the observed hierarchical interrelationships in vivo (in yeast) and in vitro (in E.coli), which are discussed. In addition, specific changes in the association of several ribosome biogenesis factors to the mutant preribosomes were observed. While in some cases the recruitment of several biogenesis factors to the preribosomes was disturbed, evidences for an inhibited release of others were provided. Altogether these results can contribute to a better understanding of the interplay between r-protein assembly events and the transient association of ribosome biogenesis factors. In addition, information of the molecular prerequisites for several maturation events, which are disturbed in the individual mutants (as pre-rRNA processing or nuclear export), can be deduced from these results
