Assembly of the eukaryotic large 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 co-transcriptional assembly of ribosomal subunits in the nucleolus is still poorly understood. Intermediates at these very early stages are extremely short-lived and transient and structures of key protein complexes that catalyze these early events have so far remained elusive. My laboratory has developed new genetic and biochemical approaches that now enable us to efficiently tag, trap and isolate early nucleolar assembly intermediates of the large 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 large ribosomal subunit and the stepwise formation of the polypeptide exit tunnel, one of its functional centers. Insights from these studies will shed light onto both the mechanisms that are employed during eukaryotic ribosome assembly to form functional centers as well as how defects in eukaryotic ribosome assembly can result in human blood disorders, which are collectively termed ribosomopathies.