Mechanisms of ribosome assembly

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 and mechanistic snapshots of assembly intermediates. Combining genetic, biochemical, mass spectrometry, and AI-based 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 co-transcriptional assembly of pre-ribosomal particles of both subunits and the subsequent transitions towards stable post- transcriptional nucleolar assembly intermediates have remained elusive due to their transient nature. This proposal describes new approaches to define the molecular mechanisms that govern co- transcriptional assembly events as well as transitions towards stable post-transcriptional assembly intermediates. My laboratory has developed new AI-based tools and biochemical approaches that now enable us to survey the entire ribosome biogenesis pathway for protein-protein interactions that guide the specific isolation of early eukaryotic ribosome assembly intermediates and study their transitions. The synergistic use of these approaches has allowed us to overcome previously intractable hurdles, thereby enabling the detailed study of essential early assembly intermediates of both ribosomal subunits. 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.