using RNAseq. Importantly, Proximity-CLIP reveals hot spots of
protein occupancy along the RNA sequence, which often relates to
elements of crucial functional and regulatory significance and facilitates studying short-lived and degradation-susceptible RNAs due
to the stabilizing effect of cross-linking to their associated RBPs.
The covalent bonds between biotin, RBPs, and RNA render
the RNP complexes resistant to stringent purification steps, maximizing the signal-to-noise ratio in the downstream highthroughput proteomic and transcriptomic analyses. The approach
is fractionation independent and allows for the isolation of compartments that are inaccessible to biochemical purification. Furthermore, the use of stringent extraction conditions promotes the
preservation of the isolated cellular components. Finally, UV crosslinking of 4-thiouridine (4SU)-labeled RNA to interacting proteins
leads to a structural change at the photoreactive nucleoside, resulting in nucleotide misincorporation during reverse transcription and
a characteristic T-to-C mutation in the corresponding cDNA
libraries. This feature allows for efficient computational removal
of contaminating sequences derived from non-cross-linked fragments of abundant cellular RNAs, further increasing the specificity
of Proximity-CLIP by reducing the false-positive detection rate.
The workflow of Proximity-CLIP comprises the following steps
(Fig. 1): (1) 4SU labeling of RNAs in living cells expressing specifically localized APEX2 (engineered ascorbate peroxidase); (2) biotinylation of APEX2-proximate proteins by incubation of cells (see
Note 1) with biotin-phenol, followed by activation of the peroxidase reaction with hydrogen peroxide (H 2 O 2 ) for 1 min, and
reaction quenching using sodium ascorbate, Trolox, and sodium
azide; (3) in vivo cross-linking of RNA and proteins using UVA or
UVB light (λ > 310 nm) during the quenching step; and (4) isolation of localized, biotinylated, and cross-linked ribonucleoprotein
(RNP) complexes by affinity chromatography.
In summary, Proximity-CLIP allows for (1) the determination
of the localized proteome in general and the RBPome in particular
using mass spectrometry; (2) the profiling of localized transcripts
using RNA-seq; and (3) the identification and quantification of
RBP-occupied cis-acting elements on transcripts, by isolation of
RNase-resistant footprints that are converted into next-generation
sequencing-compatible cDNA libraries.
2 Materials
2.1 Cells and Basic
Culture Media
This chapter can be adapted to any adherent mammalian cell line
and its growth medium. In addition to the cells expressing APEX2
targeted to the compartment of interest, cells expressing APEX2 in
a control compartment, as well as the parental cell line, which does
not express APEX2, are required (see more on cell line selection and
cell types in Note 1).
Proximity-CLIP
285
protein occupancy along the RNA sequence, which often relates to
elements of crucial functional and regulatory significance and facilitates studying short-lived and degradation-susceptible RNAs due
to the stabilizing effect of cross-linking to their associated RBPs.
The covalent bonds between biotin, RBPs, and RNA render
the RNP complexes resistant to stringent purification steps, maximizing the signal-to-noise ratio in the downstream highthroughput proteomic and transcriptomic analyses. The approach
is fractionation independent and allows for the isolation of compartments that are inaccessible to biochemical purification. Furthermore, the use of stringent extraction conditions promotes the
preservation of the isolated cellular components. Finally, UV crosslinking of 4-thiouridine (4SU)-labeled RNA to interacting proteins
leads to a structural change at the photoreactive nucleoside, resulting in nucleotide misincorporation during reverse transcription and
a characteristic T-to-C mutation in the corresponding cDNA
libraries. This feature allows for efficient computational removal
of contaminating sequences derived from non-cross-linked fragments of abundant cellular RNAs, further increasing the specificity
of Proximity-CLIP by reducing the false-positive detection rate.
The workflow of Proximity-CLIP comprises the following steps
(Fig. 1): (1) 4SU labeling of RNAs in living cells expressing specifically localized APEX2 (engineered ascorbate peroxidase); (2) biotinylation of APEX2-proximate proteins by incubation of cells (see
Note 1) with biotin-phenol, followed by activation of the peroxidase reaction with hydrogen peroxide (H 2 O 2 ) for 1 min, and
reaction quenching using sodium ascorbate, Trolox, and sodium
azide; (3) in vivo cross-linking of RNA and proteins using UVA or
UVB light (λ > 310 nm) during the quenching step; and (4) isolation of localized, biotinylated, and cross-linked ribonucleoprotein
(RNP) complexes by affinity chromatography.
In summary, Proximity-CLIP allows for (1) the determination
of the localized proteome in general and the RBPome in particular
using mass spectrometry; (2) the profiling of localized transcripts
using RNA-seq; and (3) the identification and quantification of
RBP-occupied cis-acting elements on transcripts, by isolation of
RNase-resistant footprints that are converted into next-generation
sequencing-compatible cDNA libraries.
2 Materials
2.1 Cells and Basic
Culture Media
This chapter can be adapted to any adherent mammalian cell line
and its growth medium. In addition to the cells expressing APEX2
targeted to the compartment of interest, cells expressing APEX2 in
a control compartment, as well as the parental cell line, which does
not express APEX2, are required (see more on cell line selection and
cell types in Note 1).
Proximity-CLIP
285