Assembly of the eukaryotic small ribosomal subunit

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Project Summary Ribosomes are molecular machines composed of ribosomal RNAs and up to 80 ribosomal proteins. These large assemblies catalyze protein synthesis in all cells. The long-term goal of this project is to understand how eukaryotic ribosomes are assembled with the help of more than 200 non-ribosomal factors as a series of molecular snapshots of assembly intermediates. Combining genetic, biochemical and mass spectrometry approaches with cryo-EM is an essential step to engineer, trap, isolate and determine atomic-resolution molecular snapshots of transient assembly intermediates of ribosomal subunits. Eukaryotic ribosome assembly can be subdivided into four stages, co-transcriptional assembly events and initial maturation of small and large ribosomal subunit precursors in the nucleolus, nuclear maturation of pre-40S and pre-60S particles, nuclear export, and cytoplasmic maturation. While late events in eukaryotic ribosome assembly are relatively well characterized, the early assembly of ribosomal subunits in the nucleolus is still poorly understood. Especially the mechanisms responsible for early human small ribosomal subunit assembly have remained elusive due to a biomolecular condensate that retains these early particles in human nucleoli. This proposal describes new approaches to define the molecular mechanisms that govern nucleolar maturation of the eukaryotic small ribosomal subunit. My laboratory has developed new human genome editing and biochemical approaches that now enable us to efficiently tag and isolate early nucleolar assembly intermediates of the human small ribosomal subunit. The synergistic use of these approaches has allowed us to overcome previously intractable biochemical hurdles, thereby enabling the detailed study of essential early assembly intermediates of the small ribosomal subunit. Insights from these studies will shed light onto both the mechanisms that are employed during eukaryotic ribosome assembly to coordinate key processing events as well as how defects in eukaryotic ribosome assembly can result in human blood disorders, which are collectively termed ribosomopathies.