CRISPR RiPCA for Investigating eIF4E-m7GpppX Capped mRNA Interactions

Abstract

Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m⁷GpppX cap present at the 5' terminus of coding mRNAs, with an m⁷G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technology's potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.

Figure 1.

Regulation of the epitranscriptome by RNA-binding proteins (RBPs). (A) Select examples of RNA modifications on an mRNA transcript. (B) Reader RBPs regulate many aspects of mRNA biology.

Figure 2.

Live-cell detection of RNA-protein interactions (RPIs). (A) Previous work: RNA-interaction with Protein-mediated Complementation Assay (RiPCA) utilizing transfection of LgBiT-tagged RBP plasmid and RNA substrate. (B) This work: CRISPR RiPCA using endogenously LgBiT-tagged RBP.

Figure 3.

RiPCA 2.0 for eIF4E. (A) RNA sequences used for eIF4E RiPCA. (B) Characterization of RNA binding using an eIF4E fluorescence polarization assay. (C) Chemiluminescence signal generated in SmBiT-HaloTag cells transfected with LgBiT-eIF4E (2 ng/well) and m⁷GTP RNA substrates (100 nM). (D) Lack of specificity as determined by performing RiPCA using negative control RNAs. (E) Biochemical confirmation of the specificity of LgBiT-eIF4E binding to a m⁷GTP RNA RiPCA substrate.

Figure 4.

CRISPR RiPCA for eIF4E. (A) Assay signal following delivery of SmBiT-labeled RNA by several commercial transfection reagents. (B) Titration of SmBiT-labeled m⁷GTP RNA and negative control RNAs.

Figure 5.

eIF4E CRISPR RiPCA with cap-competitive inhibitors. (A) Structures of compounds tested. (B) CRISPR RiPCA data after 6-h treatment. IC₅₀ values as 95% confidence intervals: 1 (14–36 μM), 2 (91–171 nM), and 3 (57–92 nM).

Figure 6.

eIF4E CRISPR RiPCA activity by cap-dependent pathway inhibitory compounds. (A) Mechanisms of the regulation of eIF4E and cap-dependent translation initiation. CRISPR RiPCA data after 6-h treatment with (B) eIF4E PPI modulators; IC₅₀ values as 95% confidence intervals: rapamycin (1.9–11 μM) and 4EGI-1 (1.6–3.6 μM); or (C) inhibitor of eIF4E phosphorylation (negligible effect).