Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice

Paulino Barragán-Iglesias,Michael D Burton,Vandita D Bhat,Salim Megat,Zachary T Campbell,Theodore J Price,Tzu-Fang Lou

doi:10.1038/s41467-017-02449-5

Paulino Barragán-Iglesias, Michael D Burton + Show 5 more

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https://doi.org/10.1038/s41467-017-02449-5

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Journal: Nature Communications	Publication Date: Jan 2, 2018
Citations: 138	License type: open-access

Affiliation: The University of Texas at Dallas

Abstract

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3′ end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

Highlights

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals
Our experiments focus on the major cytoplasmic Poly(A)-binding protein (PABP) isoform PABPC1 as it is the most abundant isoform based on high-throughput sequencing of the dorsal root ganglia (DRG) (Supplementary Fig. 1a)[21]
We examined the specificity of PABP for all possible 10 base sequences using in vitro selection, high-throughput sequencing of RNA, and sequence specificity landscapes (SEQRS; Fig. 1a)

Summary

Introduction

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain These results suggest that PABP is integral for nociceptive plasticity. As a proof of concept, we examine the specificity of PABP using functional genomics to probe specificity in an unbiased way Based on this information, we generate and characterize a chemically stabilized RNA substrate that binds to PABP with high specificity in vitro and impairs nascent translation in a PABPdependent mechanism in cells. The Poly(A) SPOT-ON impairs pain sensitization in multiple models of tissue injury in vivo These experiments provide a guide for the rational design of RNAbinding protein inhibitors for mechanistic studies in cells or living animals

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