Chapter 17
Proximity-CLIP Provides a Snapshot of Protein-Occupied
RNA Elements at Subcellular Resolution
and Transcriptome-Wide Scale
Daniel Benhalevy and Markus Hafner
Abstract
The distribution of messenger RNAs (mRNAs) to specific subcellular locations has been studied for the past
two decades. Technically, studies of RNA localization are lagging those related to protein localization. Here
we provide a detailed protocol for Proximity-CLIP, a method recently developed by our group, that
combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced proteinRNA cross-linking to simultaneously profile the proteome including RNA-binding proteins (RBPs) and
the RBP-bound transcriptome in any given subcellular compartment. The approach is fractionation
independent and also enables studying localized RNA-processing intermediates, as well as the identification
of regulatory cis-acting elements on RNAs occupied by proteins in a cellular compartment-specific manner.
Key words RNA-protein interactions, RNA localization, RNA regulatory elements, RNA-processing
intermediates, Subcellular RNA biology
1 Introduction
The location of molecular factors within the cell is crucial for their
regulation and function [1–3]. Excellent tools allow highresolution mapping of proteins within cells, where fractionationindependent approaches, such as fluorescence microscopy and
more recently also proximity labeling coupled to high-throughput
proteomics, profiled the localized proteome at subcellular resolution [4–9]. In contrast, studies comprehensively describing subcellular resolution data for RNA remain limited. Some of the reasons
for this include the following: (1) RNAs are often short lived due to
degradation or to maturation by sequential cleavage events.
(2) Massive unregulated RNA degradation occurs upon cell lysis
by released cellular RNases, complicating fractionation-based
approaches. (3) Imaging of RNA in intact cells is limited by sensitivity, specificity, and bias. (4) Next-generation sequencing of RNA
subspecies varying in size or in 5
0 or 3
0 features requires distinct
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_17, © Springer Science+Business Media, LLC, part of Springer Nature 2020
283
Proximity-CLIP Provides a Snapshot of Protein-Occupied
RNA Elements at Subcellular Resolution
and Transcriptome-Wide Scale
Daniel Benhalevy and Markus Hafner
Abstract
The distribution of messenger RNAs (mRNAs) to specific subcellular locations has been studied for the past
two decades. Technically, studies of RNA localization are lagging those related to protein localization. Here
we provide a detailed protocol for Proximity-CLIP, a method recently developed by our group, that
combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced proteinRNA cross-linking to simultaneously profile the proteome including RNA-binding proteins (RBPs) and
the RBP-bound transcriptome in any given subcellular compartment. The approach is fractionation
independent and also enables studying localized RNA-processing intermediates, as well as the identification
of regulatory cis-acting elements on RNAs occupied by proteins in a cellular compartment-specific manner.
Key words RNA-protein interactions, RNA localization, RNA regulatory elements, RNA-processing
intermediates, Subcellular RNA biology
1 Introduction
The location of molecular factors within the cell is crucial for their
regulation and function [1–3]. Excellent tools allow highresolution mapping of proteins within cells, where fractionationindependent approaches, such as fluorescence microscopy and
more recently also proximity labeling coupled to high-throughput
proteomics, profiled the localized proteome at subcellular resolution [4–9]. In contrast, studies comprehensively describing subcellular resolution data for RNA remain limited. Some of the reasons
for this include the following: (1) RNAs are often short lived due to
degradation or to maturation by sequential cleavage events.
(2) Massive unregulated RNA degradation occurs upon cell lysis
by released cellular RNases, complicating fractionation-based
approaches. (3) Imaging of RNA in intact cells is limited by sensitivity, specificity, and bias. (4) Next-generation sequencing of RNA
subspecies varying in size or in 5
0 or 3
0 features requires distinct
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_17, © Springer Science+Business Media, LLC, part of Springer Nature 2020
283