Preview

PDF, English (PhD thesis) - main document Download (3MB) | Terms of use

Abstract

Ribosomes are conserved in the three domains of life, Archaea, Bacteria and Eukarya. They are essential for protein synthesis in all living cells. Formation of ribosomes in eukaryotic cells occurs through an energy-consuming multi-step process, involving a myriad of ribosome assembly factors and snoRNAs. This complex process starts in the nucleolus by the synthesis of a large ribosomal RNA precursor, the 35S pre-rRNA in yeast, which is co-transcriptionally cleaved to separate the pre-40S and the pre-60S pathways. The first intermediate formed during the early phase of ribosome assembly is the 90S pre-ribosome, the precursor to the small subunit. While the 90S to pre-40S route proceeds with its own set of assembly factors and snoRNAs including U3 and U14, little is known about the formation of the earliest pre-60S intermediates and whether snoRNAs have a role in this process. To gain insight into these early steps of large subunit synthesis, I isolated and studied a pre-60S assembly intermediate at the beginning of its construction, where specific snoRNAs also come into play. This analysis revealed a network of assembly factors and snoRNAs participating in the building of this primordial pre-60S particle. This particle is characterized by two large α-solenoid scaffold modules Rrp5 and Urb1, SPOUT methyltransferases Upa1 (YGR283C) and Upa2 (YMR310C), several RNA helicases including Mak5 and two prominent snoRNAs, C/D box snR190 and H/ACA box snR37. My findings suggest that snR190 and snR37 play a structural role in pre-60S assembly and their disruption caused changes in the primordial pre-60S composition. Moreover, I used the uncharacterized Upa1 and Upa2 as single baits to isolate the primordial pre-60S, which helped to unravel its overall shape by negative stain EM. By using a dominant mak5 helicase mutant, I showed that this nascent pre-60S intermediate does not efficiently separate from the 90S pre-ribosome, which enabled for the first time the visualization of a 70 nm super 90S–pre-60S bipartite particle by electron microscopy. These findings are relevant for human ribosome biogenesis, which occurs predominantly via the post-transcriptional mechanism and, thus, is expected to also involve a pre-ribosomal intermediate carrying both 90S and pre-60S moieties before pre-rRNA cleavage. Altogether, this work sheds further light on the earliest steps of pre-60S assembly and its connection to the upstream 90S pathway.