Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments

Abstract

Methods to systematically study subcellular RNA localization are limited and lagging behind proteomic tools. Here, we combined APEX2-mediated proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced crosslinking to simultaneously profile the proteome, as well as the transcriptome bound by RNA-binding proteins in any given subcellular compartment. Our approach is fractionation-independent and enables to study the localization of RNA processing intermediates, as well as the identification of regulatory RNA cis-acting elements occupied by proteins in a cellular compartment-specific manner. We applied Proximity-CLIP to study RNA and protein in the nucleus, cytoplasm and at cell-cell interfaces. Among other insights, we observed frequent transcriptional readthrough continuing for several kilobases downstream of the canonical cleavage and polyadenylation site and a differential RBP occupancy pattern for mRNAs in the nucleus and cytoplasm. Surprisingly, mRNAs localized to cell-cell interfaces often encoded regulatory proteins and contained protein-occupied CUG sequence elements in their 3’ untranslated region.