Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.

Abstract

The processing, membrane targeting and folding of newly synthesized polypeptides is closely linked to their synthesis at the ribosome. A network of enzymes, chaperones and targeting factors engages ribosome-nascent chain complexes (RNCs) to support these maturation processes. Exploring the modes of action of this machinery is critical for our understanding of functional protein biogenesis. Selective ribosome profiling (SeRP) is a powerful method for interrogating co-translational interactions of maturation factors with RNCs. It provides proteome-wide information on the factor's nascent chain interactome, the timing of factor binding and release during the progress of translation of individual nascent chain species, and the mechanisms and features controlling factor engagement. SeRP is based on the combination of two ribosome profiling (RP) experiments performed on the same cell population. In one experiment the ribosome-protected mRNA footprints of all translating ribosomes of the cell are sequenced (total translatome), while the other experiment detects only the ribosome footprints of the subpopulation of ribosomes engaged by the factor of interest (selected translatome). The codon-specific ratio of ribosome footprint densities from selected over total translatome reports on the factor enrichment at specific nascent chains. In this chapter, we provide a detailed SeRP protocol for mammalian cells. The protocol includes instructions on cell growth and cell harvest, stabilization of factor-RNC interactions, nuclease digest and purification of (factor-engaged) monosomes, as well as preparation of cDNA libraries from ribosome footprint fragments and deep sequencing data analysis. Purification protocols of factor-engaged monosomes and experimental results are exemplified for the human ribosomal tunnel exit-binding factor Ebp1 and chaperone Hsp90, but the protocols are readily adaptable to other co-translationally acting mammalian factors.