A Comprehensive Roadmap of the Mammalian Ribo-interactome Connects Metabolism Enzymes with Specialized ER-Ribosomes

The ribosome has considerably increased in size during metazoan evolution in the form of an RNA shell that could serve as a platform for yet unknown protein interactions. Here, we have comprehensively identified the mammalian ‘ribo-interactome’ by establishing a ribosome affinity purification method. Our findings reveal a multitude of novel ribosome interacting factors, encompassing unanticipated functional categories including energy metabolism, cell redox homeostasis, as well as key protein and RNA modifying enzymes. These findings led us to characterize ufmylation, a novel posttranslational modification on ribosomes, and define its substrates. We further show that pyruvate kinase, a key enzyme for stem cell and cancer metabolism, is an RNA binding protein that unexpectedly interacts with specialized sub-pools of ribosomes at the endoplasmic reticulum (ER) and coordinates the localization and translation of ER destined mRNAs. Collectively, these studies uncover that the ribo-interactome imbues a new layer of regulatory potential in translating the genome. Overall design: Examination of ribosome footprints and RNA expression in mESCs upon PKM knockdown and of PKM RNA binding in mESCs