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Abstract
The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein – rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5′ end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.
Highlights
The structural constituents of the two eukaryotic ribosomal subunits are 4 ribosomal RNAs, namely the 25S, 18S, 5.8S and 5S rRNA and more than 79 ribosomal proteins (r-proteins)
In order to analyse in vivo functions of eukaryotic large ribosomal subunit (LSU) r-proteins we created 26 yeast mutant strains conditional for expression of individual LSU r-protein genes
All 26 mutant strains could be cultivated in galactose containing medium but stopped growth when plated on medium with glucose as the sole carbon source, indicating that each of the corresponding LSU r-proteins is essential for yeast growth
Summary
The structural constituents of the two eukaryotic ribosomal subunits are 4 ribosomal RNAs, namely the 25S, 18S, 5.8S and 5S rRNA and more than 79 ribosomal proteins (r-proteins). They assemble in a highly dynamic process that starts with the synthesis of the precursor of 25S-, 18S- and 5.8S rRNA by RNA polymerase I and initial maturation events in the nucleolus, proceeds in the nucleoplasm and ends after nucleocytoplasmic translocation of the subunits in the cytoplasm. Biochemical and bioinformatic analysis identified more than 150 protein factors and more than 70 small nucleolar RNAs involved in eukaryotic ribosome biogenesis. The association of many of these factors with different pre- 40S or pre-
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