Molecular mechanism of recognizing individual stalled ribosomes by the E3 ligases Mag2 and Fap1

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For numerous reasons the translation of mRNA by ribosomes can slow down or even stall during all phases of translation. These stalled ribosomes need to be recognized in order to elicit stress responses, degrade the faulty translation components and products, and trigger downstream quality control pathways. Several biochemical and structural studies revealed that trailing ribosomes that collide with the stalled ribosome provide a readily recognizable proxy, i.e., ribosomal collisions, which turned out to be a key signal in bacteria and eukaryotes. However, stalled initiating ribosomes and ribosomes stalling on mRNAs with low initiation efficiency are unlikely to result in collisions and require the specific recognition of individual stalled 80S ribosomes. Here, similar to collision recognition, E3 ubiquitin ligases, such as RNF10 in human cells or Mag2 and Fap1 in yeast, were recently discovered to play a central role in the recognition of such individual stalled ribosomes which are then tagged by ubiquitination of specific ribosomal proteins for downstream processes. However, the underlaying molecular mechanisms of these recognition and modification activities are not known. Therefore, the overall goal of this project is to elucidate the molecular mechanism of quality control pathway initiation by yeast E3 ligases Mag2 and Fap1 and their human homologs RNP10 and NFX1. Specifically, we suggest to address the following main questions: What features of a slow ribosome are recognized by Mag2 to perform uS3 mono-ubiquitination and what is the structural basis for recognition and poly-ubiquitination of individual 80S monosomes by Fap1 and its cofactors? How are these individual stalled 80S ribosomes recycled? What is the mechanism of RNF10 driven recognition and ubiquitination of stalled human ribosomes and how does the Fap1 homolog NFX1 interact with the human ribosome? We suggest to use cryo-electron microscopy (cryo-EM) to provide structures of Mag2-bound and Fap1-interacting ribosomal complexes which are either reconstituted in vitro or affinity purified from yeast cells. Analogously, we suggest using structural analysis by cryo-EM to characterize the mode of interaction of RNF10 and NFX1 with stalled ribosomes. We expect to provide insights into the dynamic behavior of these complexes thereby elucidating how they function in eukaryotic cells as sensors for individual stalled ribosomes. This will allow for a molecular understanding of these central and universally conserved quality control processes.