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Nat Methods. 2018 Dec;15(12):1074-1082. doi: 10.1038/s41592-018-0220-y. Epub 2018 Nov 26.

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

Author information

1
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Disease, Bethesda, MD, USA.
2
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Disease, Bethesda, MD, USA. markus.hafner@nih.gov.

Abstract

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.

PMID:
30478324
PMCID:
PMC6289640
DOI:
10.1038/s41592-018-0220-y
[Indexed for MEDLINE]
Free PMC Article

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