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. 2018 Nov 29;23(12):3124.
doi: 10.3390/molecules23123124.

In Cellulo Protein-mRNA Interaction Assay to Determine the Action of G-Quadruplex-Binding Molecules

Rodrigo Prado Martins  1 , Sarah Findakly  2 , Chrysoula Daskalogianni  3   4 , Marie-Paule Teulade-Fichou  5 , Marc Blondel  6 , Robin Fåhraeus  7   8   9   10
Affiliations

In Cellulo Protein-mRNA Interaction Assay to Determine the Action of G-Quadruplex-Binding Molecules

Rodrigo Prado Martins et al. Molecules. .

Abstract

Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.

Keywords: EBNA1; Epstein-Barr virus (EBV); G-quadruplexes; PhenDC3; protein-mRNA interactions; pyridostatin; structure-activity relationship.

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Conflict of interest statement

The authors declare no commercial or financial conflict of interest.

Figures

Figure 1
Figure 1
(A) cartoon depicting the interaction between nucleolin (NCL) and the G4 formed in the GAr-encoding sequence of EBNA1 mRNA. This interaction is crucial for EBNA1/Epstein-Barr virus (EBV) immune evasion as it inhibits both EBNA1 mRNA translation and the production of EBNA1-derived antigenic peptides, hence limiting the production of EBNA1 to the minimal level to fulfill its essential role in maintenance and replication of the viral genome and, at the same time, allowing EBNA1 to evade the immune system. (B) If the interaction between EBNA1 G4 and NCL is compromised or lost (e.g., when EBNA1ΔGAr, a form of EBNA1 deleted for its GAr domain, is expressed), then full length EBNA1ΔGAr protein as well as EBNA1-derived antigenic peptides are expressed at a higher level, leading to recognition of EBV-infected cells by the immune system of the host. (C) Schematic depicting the principle of the proximity ligation assay (PLA) between two proteins (or two epitopes of the same protein). (D) Schematic depicting the adaptation of the PLA to visualize protein/mRNA interactions in cellulo.
Figure 2
Figure 2
Use of PLA for the study of a protein-mRNA interaction. (A) H1299 cells were transfected with EBNA1 and analyzed by RNA in situ hybridization-immunofluorescence (rISH-IF) to verify the specificity of the EBNA1-digoxigenin probe and to validate the detection of probe-mRNA complexes using a mouse anti-digoxigenin antibody. Immunocomplexes were detected using an anti-mouse Alexa Fluor® 568-conjugated secondary antibody, revealing the accumulation of EBNA1 mRNA in the nucleus. EBNA1 mRNA is depicted in red. (B) Immunofluorescence (IF) was performed in H1299 cells using a rabbit anti-nucleolin antibody to set up the appropriate conditions for detection of endogenous NCL. The expected labelling of NCL in the nucleolus was confirmed. (C) PLA in EBNA1-transfected H1299 cells using mouse anti-digoxigenin and rabbit anti-nucleolin tested in (A) and (B). Anti-rabbit and anti-mouse Ig PLA probes were used following the manufacturer’s protocol to generate PLA complexes depicted as white dots. Each dot represents an interaction between NCL and EBNA1 mRNA. (D) The EBV-transformed B-cell line B95-8 was tested for endogenous NCL-EBNA1 mRNA interaction under the same conditions used in (C). PLA uncovered this interaction in the nuclear compartment as in EBNA1-transfected H1299 cells shown in (C). Scale bars represent 10 µm.
Figure 3
Figure 3
Molecular structures of the G-quadruplex (G4) ligands pyridostatin (PDS) and PhenDC3.
Figure 4
Figure 4
PLA as a tool for the characterization of RNA-binding drugs. (A) B95-8 cells treated with the G-quadruplex (G4) ligands pyridostatin (PDS) or PhenDC3 were tested for the NCL-EBNA1 mRNA interaction using PLA. PhenDC3, but not PDS, was shown to prevent the interaction, denoting that PhenDC3 competes specifically with NCL for binding EBNA1 mRNA G4. (B) Quantitative PLA was performed to quantify the effect of PDS and PhenDC3 on NCL-EBNA1 mRNA interactions. One hundred cells from control (DMSO)-, PDS-, and PhenDC3-treated groups were imaged and the number of interactions per cell was estimated using a customized protocol in ImageJ. Upper and lower lanes depict images before and after analysis, respectively. Open circles represent the nucleus included in the analysis and red dots depict PLA complexes. Cells located in the border of images and dots found outside filtered nucleus were excluded from the analysis. (C) Histogram displaying the number of NCL-EBNA1 mRNA interactions per cell from control (DMSO)-, PDS-, and PhenDC3-treated groups. Data shown are mean ± SD of three independent experiments performed as described in (B). (D) Schematic depicting the use of PLA to evaluate the effect of PDS and PhenDC3 on the NCL-EBNA1 mRNA interactions in EBV-infected B95-8 cells. PhenDC3, but not PDS, competes with NCL for binding EBNA1 mRNA G4. One possible explanation for this difference (differential binding sites for PhenDC3 and PDS, the latter forming a ternary complex EBNA1 mRNA/NCL/PDS, whereas PhenDC3 and NCL would share a common binding site) is shown. By preventing this interaction, PhenDC3 stimulates the translation of the EBNA1 mRNA and the production of EBNA1-derived antigenic peptides for the MHC class I pathway. PDS and PDC3 mean pyridostatin and PhenDC3, respectively. Scale bars represent 10 µm.
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