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

• Published in: Nature methods Vol. 15; no. 12; pp. 1074 - 1082
• Main Authors: Benhalevy, Daniel, Anastasakis, Dimitrios G., Hafner, Markus
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-12-2018; Nature Publishing Group
• Subjects: 631/1647/2017; 631/1647/2230; 631/45/500; 631/45/612/1230; Amino acid sequence; Analysis; Binding proteins; Binding Sites; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biotinylation; Coding; Cross-Linking Reagents - chemistry; Crosslinking; Cytoplasm; Fractionation; Gene expression; Gene Expression Regulation; HEK293 Cells; Humans; Interfaces; Intermediates; Isotope Labeling; Life Sciences; Localization; Messenger RNA; Nuclei (cytology); Occupancy; Polyadenylation; Polymer crosslinking; Protein Binding; Proteins; Proteomes; Proteomics; Proteomics - methods; Proximity; Regulatory proteins; Regulatory sequences; Ribonucleic acid; RNA; RNA - genetics; RNA - metabolism; RNA processing; RNA-binding protein; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Transcription; Transcriptome
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/s41592-018-0220-y

Methods for the systematic study of subcellular RNA localization are limited, and their development has lagged behind that of proteomic tools. We combined APEX2-mediated proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced crosslinking to simultaneously profile the proteome and the transcriptome bound by RNA-binding proteins in any given subcellular compartment. Our approach is fractionation independent and allows study of 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 used our method, Proximity-CLIP, to profile RNA and protein in the nucleus, in the 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. We observed that mRNAs localized to cell–cell interfaces often encoded regulatory proteins and contained protein-occupied CUG sequence elements in their 3′ untranslated region.
Proximity-CLIP, a method that combines proximity-based protein biotinylation and UV crosslinking, profiles RNAs and ribonucleoproteins in any cellular compartment.