Time-Resolved Visualization of the Yeast Ribosome-Biogenesis by Cryo-Electron Tomography

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In this project, we would like to analyze the origins of yeast ribosome biogenesis using cryo-electron tomography (cryo-ET) and correlative light and electron microscopy (CLEM). The ribosome plays a central role in gene expression. It translates the genetic information that is transcribed within the messenger RNA (mRNA) to the proteome of the cell. The importance of the ribosome and the complexity of its structure necessitate a well-coordinated and regulated assembly. Malfunctions of this process are mostly lethal for the cell and can cause severe pathology.While the mature ribosome is well studied, the structural understanding of its biogenesis remains elusive. Eukaryotic ribosome biogenesis starts in the nucleolus with the synthesis of a precursor of the ribosomal RNA. A plethora of ribosome-assembly factors associate with this RNA precursor forming pre-ribosomal particles. We recently visualized these pre-ribosomal particles in the context of the so-called Miller trees (Neyer et al., Nature 2016). Miller trees are supramolecular structures with a ribosomal DNA (rDNA) scaffold that contain actively transcribing RNA polymerase I (Pol I) enzymes, from which the nascent ribosomal RNA (rRNA) chain is emerging and folds to form the pre-ribosomal particles. These pre-ribosomal particles are located at the end of the branches of the Miller trees and are referred to as terminal knobs. Thus, the terminal knobs of the Miller trees visualize the stepwise generation of pre-ribosomes in a quasi-sequential manner. With this application, we would like to analyze the terminal knobs that form co-transcriptionally at the 5’-end of the emerging rRNA through the stepwise addition of protein complexes. Such an analysis approach is only feasible due to the defined localization of the co-transcriptionally assembling complexes on the growing pre-rRNAs of the Miller trees. In yeast, the formation of the terminal knobs is a two-stage process: Early formed knobs will be cleaved from the nascent rRNA chain to form the small ribosomal subunit and are therefore called small subunit (SSU) processome. Later formed knobs will develop to the large ribosomal subunit and are therefore called large subunit (LSU) processome. Thus, the knob generation is a two-stage process that represents the earliest precursors of the final ribosomal subunits. Due to their size (which is approximately 6 MDa for the SSU processome), cryo-electron tomography is ideally suited for the analysis of the terminal knobs. Further on specific labeling of defined states can precisely localize distinct states and facilitate our analysis. Ultimately, this study should result in a more detailed understanding of the structure and function of the SSU and LSU processomes, and provide unprecedented insights into the fascinating mechanism of the early ribosome biogenesis in vivo.