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

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.

Fig. 1

Unbiased assessment of PABP specificity and in vivo confirmation. a The SEQRS strategy begins with in vitro transcription of a DNA library containing a T7 primer (light blue), two constant regions (Primers a and b, dark blue), and a randomized 20-mer (purple). Following in vitro transcription, the library was incubated with PABP immobilized onto magnetic resin (green). RNA–protein complexes were isolated through wash steps and the bound RNAs were reverse transcribed. The T7 promoter was reattached through incorporation into a PCR primer and the process was repeated for five rounds prior to Illumina high-throughput sequencing. b Reproducibility of SEQRS. The most abundant 120,000 sequences for SEQRS replicates have a Pearson’s correlation coefficient of 0.7. The most enriched 10-mer sequence is an adenosine homopolymer and is indicated with an arrow. c Positions of the 50 most enriched 8-mer sequences from SEQRS for either PABP (green) or random sequences (purple) were calculated across known sites of PABP association outside of the Poly(A) tail in cells. Enrichment scores were calculated based on the Mann–Whitney U test. d The area under the receiver operator curve is 0.81. e The sequence logo based on the top 300 10-mer sequences following SEQRS

Fig. 2

Characterization of the in vitro binding specificity of the Poly(A) SPOT-ON and cellular uptake. a The experimental approach for generation of PABP-depleted extracts consisted of immobilization of the PABP-interacting protein (PAIP, purple) onto resin (blue). Extracts containing PABP (green) were allowed to incubate and were aspirated resulting in loss of PABP. Cy3-labeled SPOT-ONs were added to total protein lysates and analyzed by electrophoretic mobility shift assay (EMSA). b EMSA assays. SPOT-ONs were incubated with either total protein lysate or PAIP-treated lysate and incubated at 0 °C for 40 min prior to separation by non-denaturing electrophoresis. The position of free probe and a single population of protein/RNA complex is indicated. This population is only observed in the Poly(A) SPOT-ON sample and is sensitive to PAIP depletion. The scramble SPOT-ON failed to shift a single population of proteins. c Pull-down experiments were conducted from lysates as prepared in b, but the SPOT-ON was generated with a biotin tag. Immunostaining is shown for either PABP or actin as a negative control. The Poly(A) SPOT-ON specifically associated with PABP in PABP containing lysates. d Equilibrium dissociation constants were determined by florescence anisotropy measurements of either unmodified adenosine dodecamer (blue) or the Poly(A) SPOT-ON (green). A modified version of the Michaelis–Menten equation was utilized to determine the equilibrium dissociation constants of either 261 ± 54 or 301 ± 41 μM for the 12 base unmodified or Poly(A) SPOT-ON RNAs, respectively. e Stability measurements of Cy3-labeled Poly(A) (green) or scrambled (purple) SPOT-ONs were determined in 10% FBS incubated at 37 °C and compared to a non-stabilized Poly(A) RNA (blue). f Quantification of e, percentage remaining is based on the initial intensity of RNA at time zero. n = 3. Data are plotted as mean ± s.e.m. g Cellular uptake of SPOT-ONs was determined based on imaging of U2OS cells for the Poly(A) and scrambled SPOT-ONs over time. n = 6. Data are plotted as mean ± s.e.m. h Sample data are shown for the Poly(A) SPOT-ON at time zero and after 3 h

Fig. 3

The Poly(A) SPOT-ON attenuates nascent protein synthesis. a SUnSET measurements in U2OS cells were conducted in the absence of puromycin to determine background levels of signal. Puromycin staining (green), phallodin (red), DNA (blue), and the merge between channels are arranged from top to bottom. As a positive control, puromycin and vehicle were used to determine the upper limit of translation. Both homoharringtonine (HHT) and the Poly(A) SPOT-ON robustly decrease protein synthesis, whereas the scrambled SPOT-ON failed to do so. b Quantification of a, empty boxes indicate no puromycin control, pink boxes are the positive control, blue boxed are homoharringtonine, green boxes are the Poly(A) SPOT-ON, and purple boxes are the scramble control. n = 15. c PABP overexpression rescues decreased protein synthesis caused by the Poly(A) SPOT-ON. Drug treatments consisted of either vehicle or SPOT-ON in the presence of an empty vector or overexpressed PABP. The amount of vector is indicated above the row of images. Markers are arranged as in a. d Quantification of c. n = 6. Columns represent measurements in the same manner as in b. *P < 0.05, **P < 0.01, significantly different from vehicle+puro group analyzed by one-way ANOVA followed by Bonferroni post hoc test. For all graphs shown in the figure, data are plotted as mean ± s.d

Fig. 4

The Poly(A) SPOT-ON acts on initiation phase of protein synthesis. a In the first series of experiments, test compounds (e.g., hippuristanol) are added to cells and allowed to incubate prior to blockade of elongation with emetine. After 5 min puromycin is incorporated for a brief period of time. A predicted outcome of this experiment is that the ribosomes are susceptible to effects on initiation. b In a second series of experiments, elongation is blocked prior to initiation. Ribosomes are predicted to be insensitive to initiation inhibitors owing to prior arrest at a subsequent phase of translation (elongation). c, d Order of addition is indicated for either vehicle, hippuristanol, SPOT-ON RNAs, emetine, or puromycin. All samples receive emetine at the indicated time points (a, b). As before, staining is shown from top to bottom for puromycin (green), phallodin (red), DNA (blue), or a merge. e Quantification of c, empty boxes indicate no puromycin control, pink boxes are the positive control, blue boxes are hippuristanol, green boxes are the Poly(A) SPOT-ON, and purple boxes are the scramble control. Both hippuristanol and the Poly(A) SPOT-ON possess defective translation, whereas the scramble SPOT-ON does not. n = 6. f Quantification of d, where addition of emetine prior to test compounds fails to reveal significant differences for any of the test compounds. Columns represent measurements in the same manner as in e. n = 6. *P < 0.05, **P < 0.01, significantly different from vehicle + emetine + puro group analyzed by one-way ANOVA followed by Bonferroni post hoc test. For all graphs shown in the figure, data are plotted as mean ± s.d

Fig. 5

SPOT-ONs are taken up by cultured DRG sensory neurons. Uptake of SPOT-ONs was determined based on imaging of cultured DRG neurons over time. a Scramble SPOT-ON and b Poly(A) SPOT-ONs are taken up by DRG neurons and are localized into their axons after a 3-h period. c Quantification of SPOT-ONs uptake in DRG neurons from time zero to 6 h. n = 6. Data are plotted as mean ± s.e.m.

Fig. 6

The Poly(A) SPOT-ON reduces nascent protein synthesis and axonal translation in DRG neurons. a Cultured DRG neurons are incubated with SPOT-ONs (10 μM) or homoharrintonine (50 μM) for 3 h prior to addition of puromycin (1 μM) for an additional 15 min. Incubation with Poly(A) SPOT-ON, but not scrambled SPOT-ON or vehicle, significantly reduces nascent protein synthesis in DRG neurons. Staining is shown from top to bottom for puromycin (green), peripherin (red), or a merge. b Quantification of a. n = 6. *P < 0.05, **P < 0.01, significantly different from vehicle + puro group analyzed by one-way ANOVA followed by Bonferroni post hoc test. c Cultured DRG neurons are incubated with vehicle, SPOT-ONs, or hippuristanol for 3 h followed by emetine incubation (200 μM) for 5 min and puromycin (100 μM) for an additional 5 min. Incubation with Poly(A) SPOT-ON (10 μM), but not scrambled SPOT-ON or vehicle, significantly reduces proximal axonal translation (around 20–25 μM from the cell body) in peripherin-positive DRG axons. As in a, staining is shown from top to bottom for puromycin (green), peripherin (red), or a merge. d Representative images showing distal axonal ribopuromycylation (more than 25 μM from the cell body; randomly selected) in peripherin-positive DRG axons under identical conditions as described in c. e Quantification of images in c. n = 20. *P < 0.05, **P < 0.01, significantly different from vehicle+E+P group analyzed by one-way ANOVA followed by Bonferroni post hoc test. f Quantification of images in d. n = 9. *P < 0.05, **P < 0.01, significantly different from vehicle +E + P group analyzed by one-way ANOVA followed by Bonferroni post hoc test. For all graphs shown in the figure, data are plotted as mean ± s.e.m.

Fig. 7

Binding protein PABP is present throughout the peripheral nervous system. a PABP (green) is highly expressed in cultured DRG neurons and their axons including growth cones and co-localizes with peripherin immunoreactivity, a marker for unmyelinated sensory neurons (red and merge). b PABP is broadly expressed in the majority of DRG neurons and co-localizes with peripherin and TRPV1, a nociceptive marker for both C and Aδ fibers. c PABP co-localizes with the neuronal marker NeuN and is also expressed in TRPV1-positive and IB4-positive pre-synaptic endings of DRG neurons in the spinal dorsal horn. PABP is also differentially expressed in microglia (CD11b+) and astrocytes (GFAP+) in the spinal dorsal horn. As shown in the figure, 18.6 ± 1.9% of the PABP immunoreactive fibers co-localize with TRPV1, 11.3 ± 1.2% with IB4, 29.1 ± 1.7% with GFAP, and 14.8 ± 2.2% with CD11b. n = 5 slices from L4–L6 spinal dorsal horn. Data are expressed as mean ± s.e.m. d PABP present in small-diameter sensory axons containing peripherin and in Schwann cells (MPz+) in the sciatic nerve

Fig. 8

The Poly(A) SPOT-ON reduces pain sensitization in mice produced by intraplantar NGF or IL-6 administration and after plantar incision. a, b Intraplantar injection with vehicle or scrambled SPOT-ON (0.3–1 μg) did not reduce NGF-induced mechanical hypersensitivity or priming produced by intraplantar injection with PGE₂ (100 ng) at day 9 after surgery. c, d Intraplantar injection with Poly(A) SPOT-ON (1 μg) reduces NGF-induced mechanical hypersensitivity and blocked the development of PGE₂-induced hyperalgesic priming. *P < 0.05, **P < 0.01, significantly different from NGF+vehicle group analyzed by two-way ANOVA followed by Bonferroni post hoc test. e, f Intraplantar injection with vehicle or scrambled SPOT-ON (0.3–1 μg) did not reduce IL-6-induced mechanical hypersensitivity or priming produced by PGE₂. g, h Intraplantar injection with Poly(A) SPOT-ON (1 μg) reduces IL-6-induced mechanical hypersensitivity and blocked the development of PGE₂-induced hyperalgesic priming. *P < 0.05, **P < 0.01, significantly different from IL-6+vehicle group analyzed by two-way ANOVA followed by Bonferroni post hoc test. i, j Following plantar incision, local injection with Poly(A) SPOT-ON (10 μg), but not scrambled SPOT-ON (10 μg), reduces mechanical hypersensitivity, contributed to resolution of pain sensitization, and blocked development of hyperalgesic priming when animals were challenged with PGE₂ at day 15. *P < 0.05, **P < 0.01, significantly different from incision+scramble group analyzed by two-way ANOVA followed by Bonferroni post hoc test. k, l Intraplantar injection of the Poly(A) SPOT-ON, but not scrambled SPOT-ON, significantly reduces the development of paw guarding following surgery as well as PGE₂-induced priming. *P < 0.05, **P < 0.01, significantly different from incision+scramble group analyzed by two-way ANOVA followed by Bonferroni post hoc test. m, n Intraplantar injection of the Poly(A) SPOT-ON, but not scrambled SPOT-ON, significantly reduces the presence of facial grimace following surgery and after priming with PGE₂. *P < 0.05, **P < 0.01, significantly different from incision+scramble group analyzed by two-way ANOVA followed by Bonferroni post hoc test. o Paw incision significantly increases the temperature in the incised paw of mice 24 h after surgery. Under these conditions, local administration of the Poly(A) SPOT-ON, but not scrambled SPOT-ON, significantly decreased the incised paw temperature 24 h after surgery. p Quantification of incised and non-incised paw temperature from scrambled and SPOT-ON groups 24 h after surgery. *P < 0.05, **P < 0.01, significantly different from incision+scramble group analyzed by Student's t test. n = 6 per group. For all graphs showing in the figure, data are plotted as mean ± s.e.m.

Fig. 9

The Poly(A) SPOT-ON reduces pain sensitization produced by capsaicin. a The Poly(A) SPOT-ON (10 μg) inhibits the mechanical hypersensitivity produced by intraplantar capsaicin (5 μg) and b blocks the development of hyperalgesic priming. CGRP_8–37 (1 μg) has a transient antinociceptive effect at 3 h post capsaicin with no changes after the precipitation of priming with PGE₂. *P < 0.05, **P < 0.01, significantly different from scramble SPOT-ON+capsaicin (CAP) group analyzed by two-way ANOVA followed by Bonferroni post hoc test. c The Poly(A) SPOT-ON and CGRP_8–37 attenuate the thermal hypersensitivity produced by capsaicin. *P < 0.05, significantly different from Poly(A) SPOT-ON+capsaicin (CAP) group and ^& P < 0.05, significantly different from baseline (BL) analyzed by two-way ANOVA followed by Bonferroni post hoc test. Not significantly different (NS) compared to baseline (BL). d No changes in thermal hypersensitivity are detected after priming revealed by PGE₂. e The Poly(A) SPOT-ON and CGRP_8–37 block the transient increase in paw temperature produced by intraplantar capsaicin administration. **P < 0.01, significantly different from the non-injected paw or the Poly(A) SPOT-ON injected paw analyzed by one-way ANOVA followed by Bonferroni post hoc test. Not significantly different (NS) compared to non-injected paw. f No changes in paw temperature are present after priming (injected vs. non-injected paw). n = 6 per group. For all graphs shown in the figure, data are plotted as mean ± s.e.m.