In vivo proximity labeling for the detection of protein-protein and protein-RNA interactions

Abstract

Accurate and sensitive detection of protein-protein and protein-RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo. Using quantitative mass spectrometry and deep sequencing, we show that IPL correctly identifies known protein-protein and protein-RNA interactions in the nucleus of mammalian cells. Thus, IPL provides additional temporal and spatial information for the characterization of biological interactions in vivo.

Keywords: Proximity labeling; RNA-seq; biotinylation; covalent tag; protein−RNA interactions; protein−protein interactions.

Figure 1

In vivo proximity labeling (IPL). (A) Schematic depiction of the IPL strategy. A protein of interest (POI) is fused with monomeric streptavidin (mSA), which recruits a probe (bio-PA) constituted of biotin (red triangle) linked to a photoactivatable group (yellow circle). After UV irradiation the photoactivatable group reacts with proteins and other macromolecules that are in close proximity to the POI in vivo. Because bio-PA is now covalently bound to putative POI interactors, the cells can be lysed under harsh conditions and the identity of the POI interactors can be revealed by streptavidin purification followed by mass spectrometry. (B) Chemical structure of the bio-ASA heterobifunctional probe used for IPL.

Figure 2

Self- and trans-labeling by IPL in the PRC2 complex. (A) Schematic depiction of self-labeling reactions (left) and trans-labeling reaction (right) in the context of the PRC2 complex used for this proof of concept. (B) 293T-REx cells expressing the N-terminal fusions mSA–EZH2 or GAL4–EZH2 (negative control) were subjected to IPL with the indicated concentrations of bio-ASA. Biotinylated proteins were recovered by streptavidin pull-down and revealed by western blots with EZH2 and EED antibodies. (C) IPL of GAL4–EZH2 (control) and mSA–EZH2 followed by IP for PRC2 components (EZH2, EED, and SUZ12) as well as a PRC1-associated factor (SCML2) as an additional control. Western blots for EZH2, EED, and SCML2 (left) and biotin (right) are shown.

Figure 3

Unbiased IPL proteomics. (A) 293T-REx expressing mSA–EZH2 or GAL4–EZH2 were subjected to IPL. Biotinylated proteins were purified using streptavidin and identified by MS. Each identified protein is represented by a dot in the scatter plot. The x axis indicates the normalized and log-converted average of unique peptide abundance in mSA–EZH2 and GAL4–EZH2; the y axis indicates the specific enrichment in mSA–EZH2 samples (above the dotted line) compared to the GAL4–EZH2 control. Red dots indicate the position of the PRC2 core components. Data is averaged from 3 biological replicates. (B) Scatter plot with SILAC enrichment scores for polypeptides biotinylated by IPL in mSA–EZH2 cells. The two axes indicate the normalized ratios of spectral counts for each polypeptide in the heavy sample vs the light sample (H/L) in the forward SILAC (GAL4–EZH2 light, mSA–EZH2 heavy) and reverse SILAC (GAL4–EZH2 heavy, mSA–EZH2 light).

Figure 4

IPL of protein-interacting noncoding RNAs. (A) Schematic depiction of the labeling reaction. The mSA tag was fused to the N terminus of SNRNP70, which interacts with the U1 spliceosomal snRNA. After IPL, part of the label is deposited on the RNA. (B) IPL using a different concentration of bio-ASA probe (x axis) was performed on 293T-REx expressing either mSA–SNRNP70 (black squares) or FH–SNRNP70 (white circles) as a control. RNA was extracted with TRIzol and precipitated with streptavidin-coupled magnetic beads. The y axis shows the enrichment of U1 RNA compared to 5S rRNA after precipitation, as determined by RT-qPCR. (C) Enrichment of U1 RNA in SNRNP70 IPL, as determined by deep sequencing and mapping to annotations in ENSEMBL 71. Mean abundance is plotted on the x axis, and input-corrected enrichment is plotted on the y axis. U1 and U2 genes are highlighted in red and blue, respectively. Data is from 2 biological replicates. (D) Quantification of U1 and U2 IPL enrichment according to deep sequencing. Reads per kilobase per million (RPKM) for each U1 and U2 locus were calculated in FH and mSA IPLs and divided for the RPKM of the respective genes in the input RNA. Bars represent mean + SEM.