The ribosome, the protein factory of the cell, is essential for all life forms. The ribosome is a large RNA-protein machine. It is built in a complex, multi-step process that involves a large number of accessory trans-acting factors and the synthesis consumes a considerable part of the cellular energy. The ribosomal RNA is transcribed as a large precursor rRNA (pre-rRNA) molecule that undergoes extensive processing during maturation, including chemical modifications, pre-rRNA cleavage events, pre-rRNA folding and assembly with ribosomal proteins. More than 200 non-ribosomal proteins and small nucleolar RNAs ensure a successful maturation of the two ribosomal subunits during a pathway that starts with coupled synthesis and processing of the pre-rRNA within the nucleolus. Processing continues through the nucleus and ends with the final maturation in the cytoplasm.

We have studied one of the eukaryotic ribosomal biogenesis proteins, Mrd1 to learn about its essential function in the pre-ribosome maturation process in the yeast, Saccharomyces cerevisiae. Mrd1 contains multiple RNA-binding domains and the protein and its modular design is conserved throughout eukarya. Evolution of Mrd1 is most likely coupled to a common eukaryotic way of producing ribosomes. Together with a large set of other factors, Mrd1 associates early with the nascent pre-rRNA and forms a 90S pre-ribosome that can be seen in Chromatin Miller spreads of active rRNA genes as large terminal knob structures on the growing pre-rRNA. In the absence of Mrd1, essential steps in pre-ribosome maturation cannot occur and small ribosomal subunits are not produced. We have demonstrated that Mrd1 interacts with the pre-rRNA in vivo at two specific sites within the 18S rRNA sequence, both located close in space to where the essential and universally conserved central pseudoknot of the small ribosomal subunit is formed. Furthermore, we have shown that Mrd1 influences the release of the U3 snoRNA from the pre-ribosome. U3 snoRNA is essential for synthesis of the small ribosomal subunit and is involved in pseudoknot formation. Our results show that Mrd1 is present within the pre-ribosome at a crucial location and that it is required for essential maturation steps. Based on our results, we hypothesize that Mrd1 modulates the pre-rRNA folding and assembly to assist pre-ribosome structures necessary for pseudoknot formation and early cleavages. This essential function is conserved in all eukaryotes. 

Ribosomes are macromolecular machines that are responsible for production of every protein in a living cell. Yet we do not know the details about how these machines are formed. The ribosome consists of four RNA strands and roughly 80 proteins that associate with each other in the nucleolus and form pre-ribosomal complexes. Eukaryotes, in contrast to prokaryotes, need more than 200 non-ribosomal factors to assemble ribosomes. These associate with pre-ribosomal complexes at different stages as they travel from the nucleolus to the cytoplasm and are required for pre-rRNA processing. We do however lack knowledge about the molecular function of most of these factors and what enables pre-rRNA processing. Especially, information is missing about how non-ribosomal factors influence folding of the pre-rRNA and to what extent the pre-ribosomal complexes are restructured during their maturation. 

This thesis aims to obtain a better understanding of the earliest events of ribosome assembly, namely those that take place in the nucleolus. This has been achieved by studying the essential protein Mrd1 by mutational analysis in the yeast Saccharomyces cerevisiae as well as by obtaining structural information of nucleolar pre-ribosomal complexes. Mrd1 has a modular structure consisting of multiple RNA binding domains (RBDs) that we find is conserved throughout eukarya. We show that an evolutionary conserved linker region of Mrd1 is crucial for function of the protein and likely forms an essential module together with adjacent RBDs. By obtaining structural information of pre-ribosomal complexes at different stages, we elucidate what structuring events occur in the nucleolus.  We uncover a direct role of Mrd1 in structuring the pre-rRNA in early pre-ribosomal complexes, which provides an explanation for why pre-rRNA cannot be processed in Mrd1 mutants.