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

Rodrigo Prado Martins,Marie-Paule Teulade-Fichou,Robin Fåhraeus,Sarah Findakly,Marc Blondel,Chrysoula Daskalogianni

doi:10.3390/molecules23123124

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.

Highlights

Accumulating evidence indicates an ever-expanding role for RNAs in regulating most aspects of cell biology that range from small non-coding RNAs to messenger RNAs
We have recently reported that nucleolin (NCL) directly binds G4 formed in the GAr-encoding sequence of the Epstein Barr virus (EBV) EBNA1 messenger RNA (mRNA) (Figure 1A,B, see Reference [10])
GAr-coding sequence of the EBNA1 mRNA and this is essential for suppressing the translation of the EBNA1 mRNA and the production of antigenic peptides (Figure 1A,B)

Summary

Introduction

Accumulating evidence indicates an ever-expanding role for RNAs in regulating most aspects of cell biology that range from small non-coding RNAs to messenger RNAs (mRNAs). The more traditional role of mRNAs as “only messengers” is changing and new knowledge is emerging showing how the encoding sequences are taking on more diverse functions and can influence the activity of the encoded proteins. If not all aspects of mRNAs, and this is presumably true for non-coding. RNAs, are governed by interactions with cellular proteins. These ribonucleoproteins (RNP) complexes control RNA metabolism and form scaffolds to orchestrate and organize protein networks and complex functional units [1]. Interfering with specific protein-RNA complexes holds promise for new therapeutic developments as well as furthering our understanding of cell biological process.

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