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.

RNA-protein interaction mapping via MS2 or Cas13-based APEX targeting

“…While our analyses demonstrate the high specificity achieved by the proteomic experiment, we find that our sensitivity is limited; only one (DKC1) of the six core components of telomerase (Supplementary Table 1) was identified. Of the remaining five proteins, four were not detected at all by the mass spectrometer in our experiment and were similarly undetected in previously published APEX nuclear proteomes [56][57][58] , suggesting that they are likely low-abundance proteins (e.g. TERT 27 ) or lack surface-exposed tyrosines for APEX biotinylation.…”

Genomic profiling of PML bodies reveals transcriptional regulation by PML bodies through the DNMT3A exclusion

“…APEX2 is an engineered 38 peroxidase that displays superior activities in living cells 14 , where it catalyses the oxidization of 39 biotin-phenol to generate a radical form in an H2O2-dependent manner and results in 40 biotinylation of proteins in tight spaces (< 20 nm in radius). APEX2 has been used for the 41 identification of proteins proximal to target proteins or RNA by being combined with mass 42 spectrometry [15][16][17][18] . Compared with other proximity labelling techniques for chromatin studies 43 such as bioChIP-seq 19 , imuno-Trap 20 and TSA-seq 21 , the quick reaction (1 min) by APEX2 in 44 living cells is suitable to address the dynamic association between nuclear domains and 45 chromatin.…”

Proximity labeling to detect RNA–protein interactions in live cells

“…Almost no discernible signal is seen between the PUF RNA motifs and the scrambled controls. Based on what we have found in the literature, APEX2 can identify proximate associations, although only with appropriate controls (not unlike birA*) and with SILAC labeling (unnecessary for birA*) [37][38][39][40]. BirA* is the predominant method used to identify candidate protein-protein interactions [32,34,35,41].…”