The emergence of numerous diseases caused by defective assembly of ribosomes calls for a deeper understanding of human ribosome biogenesis. Yet, in human cells the assembly of ribosomal subunits is a vital process of daunting complexity, which requires several hundreds of assembly factors (AFs). Together with small nucleolar RNAs (snoRNAs) these factors facilitate a cascade of modification, processing and folding events of the ribosomal RNA (rRNA) that are tightly coordinated with the incorporation of a large set of ribosomal proteins between the nucleolus and the cytoplasm. This process has been extensively studied in fungal model organisms such as S. cerevisiae, however, assembly and rRNA processing pathways differ significantly between yeast and mammals. Many unique AFs have been identified in humans and several diseases, so-called ribosomopathies, have been assigned to ribosome biogenesis defects. Yet, so far only a single publication (Ameismeier et al., 2018) provides direct molecular information on the structural basis and architectural transitions of human ribosome maturation. Therefore, I propose to provide a near complete structural inventory of human ribosome biogenesis by using state-of-the-art cryo-electron microscopy (cryo-EM) on purified native pre-ribosomal particles. Corresponding analysis by chemical cross-link mass spectrometry, Nanopore sequencing and cryo-tomography as well as functional assays probing for nuclear export and RNA processing defects will gain complementary functional information. Finally, shot-gun cryo-EM of total pre-40S, pre-60S and 90S intermediates will be established in order to quantitatively characterize the equilibrium flow of ribosome assembly in normal and challenged human cells. Together, these insights will provide the basis for a mechanistic understanding of human ribosome biogenesis and will thereby lay the foundation for better relating this process to regulatory pathways and disease.
Field of science
- /natural sciences/biological sciences/genetics and heredity/rna
- /natural sciences/biological sciences/molecular biology/structural biology
- /natural sciences/chemical sciences/analytical chemistry/mass spectrometry
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