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Abstract

Ribosomes are the cellular machines that are built from proteins and rRNA and are responsible for de novo protein production. Given the essentiality of this process and the need to be tightly controlled and regulated, the production of ribosomes, or ribosome biogenesis, has to be executed flawlessly. Among cellular processes conserved from prokaryotes and eukaryotes, ribosome biogenesis stands out as herculean task: in eukaryotes, over 200 assembly factors (AFs) help navigating the 18S, 25S and 5.8S rRNA species along their unique pathway from one common precursor to mature rRNA molecules, which are complexed with ribosomal proteins and ready to engage in efficient and faultless de novo production of proteins. Despite genetic, biochemical and structural research over several decades, the precise role of most eukaryotic AFs remained unknown. In my PhD thesis, I have investigated the role of the KH domain containing assembly factor Krr1 during maturation of the first biochemically accessible ribosomal pre-cursor, the 90S pre-ribosome. One key feature of the 90S particle is that its AFs function as a scaffold, which allows for the maturation of the 35S pre-rRNA organized in subdomains. These subdomains are positioned in different regions of the 90S particle (head, body and base). I generated cryo-EM structure-based mutations of different AFs with the focus on Krr1 and performed biochemical analysis of 90S particles isolated from such mutant yeast cells. Following this approach, I was able to uncover and dissect the genetic and functional network between Krr1 and several AFs, reaching from the head through the body to the base region of the 90S. Disruption of Krr1’s contact site in the base region is genetically and functionally connected to its interaction site in the head of the 90S pre-ribosome. Both are important at later stages of 90S particle maturation, before the huge precursor transitions into an early pre-40S particle. Together with the finding that maturation of the different 90S rRNA subdomains does not follow the direction of transcription (5’>3’), this places Krr1 in a special position as an assembly factor that bridges the three different regions of the 90S pre-ribosome. Thus, Krr1 is not only a placeholder for the essential 90S/pre-40S factor Pno1/Dim2, but also can act as a sensor of the coordinated assembly and maturation processes in the 90S particle “from head to toe”.