The porphyrin TMPyP4 inhibits elongation during the noncanonical translation of the FTLD/ALS-associated GGGGCC repeat in the C9orf72 gene

Abstract

GGGGCC (G₄C₂) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G₄C₂ repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G₄C₂ repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G₄C₂ repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G₄C₂ repeat RNA. Urea-resistant interaction between G₄C₂ repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G₄C₂ repeat translation elongation.

Keywords: DPR; G-quadruplex; RAN translation; elongation; frontotemporal dementia; inhibitor; microsatellite; motor neuron disease; repeat expansion; ribosome stalling.

Figure 1

TMPyP4 inhibits RAN translation in a cellular model of C9orf72 repeat expansion without affecting expression and localization of G₄C₂repeat RNA. A, schema for (G₄C₂)₈₀ repeat plasmid and repeat deletion “Del rp” plasmid. Both share multiple stop codon containing 5′ flanking region of the expanded C9orf72 G₄C₂ repeat. B and C, increasing doses of TMPyP4 significantly inhibit poly-GA expression in (G₄C₂)₈₀ transfected HeLa cells. n = 3, experiments performed in duplicates. ANOVA with Dunnett post hoc test versus “0.” ∗∗∗p < 0.0001. D, treatment with increasing doses of TMPyP4 gives modest inhibition on repeat RNA expression levels in RT-quantitative PCR. n = 2, experiments performed in duplicates. ANOVA with Dunnett post hoc test versus “0.” #p = 0.0708. E, percent of nuclear distribution of MALAT-1, GAPDH, β-actin, and G₄C₂ repeat RNA of cells cultured in the presence (+) or the absence (−) of 20 μM TMPyP4. TMPyP4 does not significantly affect the percent of nuclear distributions of these RNA. n = 3. Two-tailed paired t test. All graphs are shown as mean ± SD. Each dot represents single data point. GA, glycine alanine; G₄C₂, GGGGCC; RAN, repeat-associated non-AUG.

Figure 2

TMPyP4 inhibits RAN translation of C9orf72 expanded G₄C₂repeat in all reading frames but spares global cellular translation. A, schema of strong CMV promoter-driven (G₄C₂)₈₀ plasmids lacking ATG initiation codon. With artificial frame shift insertion before C-terminal FLAG tag, each plasmid labels one DPR (GA, GP, or GR) with FLAG tag. B and C, dot blot analysis of cells transfected with repeat plasmid (A) or mock plasmid cultured for two overnights in the presence (+) or the absence (−) of 20 μM TMPyP4. n = 3. Two-tailed paired t test. D, schematic representation of an EF1 promoter-driven EGFP expression plasmid containing conventional ATG initiation codon. E and F, no inhibition of cellular EGFP expression with increasing doses of TMPyP4. n = 3, experiments performed in duplicates. ANOVA with Dunnett post hoc test versus “0.” G, puromycin incorporation assay monitoring active cellular translation. Cells were treated with/without TMPyP4 or CHX and then pulse labeled with puromycin. Actively translating proteins during pulse labeling (i.e., proteins incorporated puromycin) were visualized with antipuromycin antibody. β-actin blot is shown as loading control. H, signal intensities of each lane of puromycin blot were measured and normalized with corresponding β-actin signals. While translation inhibitor CHX suppressed global translation, TMPyP4 significantly enhanced puromycin incorporation. n = 4, experiments performed in duplicates. ANOVA with Dunnett post hoc test versus “0” in each compound. Data points “0” in these two graphs show same data. ∗p = 0.0112 and ∗∗∗p = 0.0003. All graphs are shown as mean ± SD. Each dot represents single data point. CHX, cycloheximide; CMV, cytomegalovirus; DPR, dipeptide repeat; EF1, elongation factor 1; EGFP, enhanced GFP; GA, glycine alanine; G₄C₂, GGGGCC; GP, glycine alanine; GR, glycine arginine; RAN, repeat-associated non-AUG.

Figure 3

TMPyP4 inhibits not only non-AUG-dependent but AUG-dependent G₄C₂repeat translation in cells. A, schema for (G₄C₂)₈₀ repeat plasmid with artificial insertion of good Kozak sequence with conventional ATG initiation codon in poly-GA frame. B and C, increasing doses of TMPyP4 significantly inhibit poly-GA expression in ATG-(G₄C₂)₈₀ transfected cells. n = 4 (or three in 50 and 100 μM). Experiments performed in duplicates. ANOVA with Dunnett post hoc test versus “0.” ∗p = 0.0125 and ∗∗∗∗p < 0.0001. Graphs are shown as mean ± SD. Each dot represents single data point. GA, glycine alanine; G₄C₂, GGGGCC.

Figure 4

TMPyP4 preferentially inhibits G₄C₂repeat translation regardless of AUG initiation or non-AUG initiation in in vitro translation assay. A, formaldehyde denaturing gel electrophoresis of purified in vitro–transcribed RNA with 5′ cap and 3′ polyadenylation. B and C, Western blot analysis of samples from in vitro translation with rabbit reticulocyte lysates in the presence or the absence of increasing doses of TMPyP4. Quantifications are shown in C. Four independent experiments. One-way ANOVA with Tukey post hoc test. ∗∗p = 0.0091 (EGFP versus GA) or p = 0.0030 (EGFP versus ATG-GA). Graphs are shown as mean ± SD. Each dot represents single data point. EGFP, enhanced GFP; GA, glycine alanine; G₄C₂, GGGGCC.

Figure 5

TMPyP4 induces polysome retention on G₄C₂repeat RNA.A, schematic representation of the procedures of polysome profiling assay (see also Experimental procedures section). Cytoplasmic fractions from repeat transfected HeLa cells cultured in the presence or the absence of 20 μM TMPyP4 were loaded onto the top of 7 to 47% linear sucrose gradient followed by ultracentrifugation. Fractions (fr.) were successively isolated from the top of the gradient. B, representative polysome profiles in (G₄C₂)₈₀-transfected HeLa cells treated or not treated with TMPyP4. Fr. 1 to 3 represent nontranslating total RNA (saturating absorbance at 260-nm signal). Fr. 4 to 8 “40S + 60S + monosome” are considered to represent the mRNA containing 40S small ribosome subunit, 60S large ribosome subunit, and monosome. Fr. 9 to 13 “low-weight (LW) polysome” and fr. 14 to 22 “high-weight (HW) polysome” are estimated to be enriched in the signal from two to three (LW) or four or more (HW) ribosome containing mRNA. C, electropherograms of bioanalyzer analysis of RNA purified from fr. 4 to fr. 7 of nontreated cells and total RNA from HeLa cells. rRNA ratio (28S/18S) is automatically calculated from each electropherogram. 25-nucleotide (nt) peak represents a supplemented size marker. D, RT-quantitative PCR analysis of each of the combined 40S + 60S + monosome, LW polysome, and HW polysome fractions for G₄C₂ repeat RNA. Repeat RNA signals are normalized with spiked EGFP signals by using ΔΔCT method. Graphs are shown as mean ± SD. Each dot represents single data point. Vertical axis is shown as abundance of G₄C₂ repeat RNA (percent of total detected [= sum of (the combined 40S + 60S + monosome) + LW polysome + HW polysome]). Four independent experiments. Two-tailed paired t test. E, representative distributions of repeat RNA signals from the polysome profile of cells treated or untreated with TMPyP4. G₄C₂ repeat RNA signals are normalized with spiked EGFP RNA by using ΔΔCT method. Vertical axis is shown as percent abundance of repeat RNA (of total detected [fr. 4–22]). EGFP, enhanced GFP; G₄C₂, GGGGCC.

Figure 6

TMPyP4 induces urea-resistant electromobility shift on G₄C₂repeat RNA. A, EMSAs for FAM-labeled synthetic (G₄C₂)₈ repeat RNA premixed with increasing concentrations of TMPyP4. RNA–TMPyP4 mixtures were run on nondenaturing 10% TBE gel (upper panel) or denaturing 15% TBE gel containing 6.5 M urea (lower panel). B, EMSA for FAM-labeled generic RNA premixed with increasing concentrations of TMPyP4. The mixtures were run on nondenaturing 10% TBE gel (upper panel) or denaturing 15% TBE gel containing 6.5 M urea (lower panel). Three independent experiments. G₄C₂, GGGGCC.