RAPIDASH: Tag-free enrichment of ribosome-associated proteins reveals compositional dynamics in embryonic tissues, cancer cells, and macrophages [RNA-Seq]

Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including DHX30 and LLPH, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts. Overall design: RAPIDASH characterization by short read sequencing of polyA-selected RNAs differentially enriched by the additional sulfhydryl-charged resin chromatography step compared to sucrose cushion alone. Refer to Figure S2