TY - JOUR
T1 - Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments
AU - Benhalevy, Daniel
AU - Anastasakis, Dimitrios G.
AU - Hafner, Markus
N1 - Publisher Copyright:
© 2018, This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.
PY - 2018/12/1
Y1 - 2018/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85057318108&partnerID=8YFLogxK
U2 - 10.1038/s41592-018-0220-y
DO - 10.1038/s41592-018-0220-y
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C2 - 30478324
AN - SCOPUS:85057318108
SN - 1548-7091
VL - 15
SP - 1074
EP - 1082
JO - Nature Methods
JF - Nature Methods
IS - 12
ER -