RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats - PubMed (original) (raw)

. 2019 Sep;22(9):1383-1388.

doi: 10.1038/s41593-019-0455-7. Epub 2019 Jul 29.

Tania F Gendron 3, Teresa Niccoli 4 5 6, Naomi R Genuth 1 2 7, Rosslyn Grosely 8, Yingxiao Shi 9, Idoia Glaria 4 5, Nicholas J Kramer 1 10, Lisa Nakayama 1, Shirleen Fang 1, Tai J I Dinger 1 2, Annora Thoeng 4 5 6, Gabriel Rocha 9, Maria Barna 1 7, Joseph D Puglisi 8, Linda Partridge 6, Justin K Ichida 9, Adrian M Isaacs 4 5, Leonard Petrucelli 3, Aaron D Gitler 11

Affiliations

RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats

Shizuka B Yamada et al. Nat Neurosci. 2019 Sep.

Abstract

Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Unconventional translation (RAN translation) of C9orf72 repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia and other neurodegenerative diseases caused by nucleotide repeat expansions.

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Conflict of interest statement

Competing Interests

A.D.G. has served as a consultant for Aquinnah Pharmaceuticals, Prevail Therapeutics, and Third Rock Ventures

Figures

Figure 1:

Figure 1:. RPS25 is required for efficient RAN translation in yeast and human cells.

(a) Detection of RAN-translated DPR in yeast lysate using a poly(GP) immunoassay. Wildtype (BY4741) yeast were transformed with an empty vector or constructs expressing either 2 or 66 C9orf72 GGGGCC repeats (C9 2R or 66R) under the control of a galactose inducible promoter. DPR production was assayed in yeast lysates using a poly(GP) immunoassay. We detected poly(GP) in the C9 66R expressing yeast (two-tailed, unpaired t-test; n=3 WT and WT C9 2R transformations; n=8 independent rps25AΔ C9 66R transformations; ****p<0.0001; mean +/− s.e.m.). (b) Schematic of yeast poly(GP) and ATG-GFP counter screen to identify RAN translation regulators. C9 40R expression constructs were introduced by transformation or mating into yeast mutants from the deletion collection (MATa; non-essential genes) and DAmP library (essential genes). Mutants were assayed for poly(GP) levels using a poly(GP) immunoassay and counter-screened with a GFP immunoassay. Data provided in Table S1. (c) Fold-change poly(GP)-levels of yeast mutants compared to wildtype yeast expression is shown (n=3 independent transformations for each strain). (d) Independent validation of rps25AΔ mutant expressing C9 66R using poly(GP) immunoassay. Poly(GP) levels were approximately 50% lower in rps25AΔ compared to wildtype yeast (two-tailed, unpaired-test; n=3 independent deletion strains; ***p=0.0010, *p=0.0248; mean +/− s.e.m.). (e) Immunoassay shows RPS25KO in the human Hap1 cell line reduces poly(GP) levels (two-tailed, unpaired t-test; n=5 independent cell culture experiments; ***p=0.0002; mean +/− s.e.m.). (f) Lysates from transfected Hap1 cells were immunoblotted for poly(GA) expression (HA-epitope tag). (g) Quantification of (f) (uncropped blots for this and all subsequent blots can be found in Supplemental Fig. 11; two-tailed, unpaired t-test; n=3 independent cell culture experiments; ****p<0.0001; mean +/− s.e.m.). (h) Immunoassay shows RPS25KO in Hap1 cells reduces poly(GR) levels to that of Hap1 wildtype transfected with empty vector. Full conditions and ANOVA statistics shown in Fig. S3 (ordinary one-way ANOVA with Tukey’s multiple comparisons, n=3 independent cell culture experiments; ****p<0.0001; mean +/− s.e.m.). (i) Lysates from transfected HeLa cells were immunoblotted for poly(Q) and poly(A) ATXN2 RAN products. (j and k) Quantification of (i) where poly(Q) or poly(A) are normalized to GAPDH. (j) ATXN2 CAG108 RAN translated poly(Q) products are reduced in HeLa cells harboring a CRISPR-induced mutation that markedly reduces level of RPS25 (RPS25KD) compared to HeLa control cell (two-tailed, unpaired t-test; n=3 independent cell culture experiments; **p=0.0059, n.s., not significant p=0.0946; mean +/− s.e.m.). (k) ATXN2 CAG108 RAN poly(A) products are reduced in HeLa RPS25KD mutant compared to HeLa control (two-tailed, unpaired t-test; n=3 independent cell culture experiments; *p=0.0473; mean +/− s.e.m.). Additional statistical details for this figure and subsequent figures are provided in Table S4 and the Methods.

Figure 2:

Figure 2:. RPS25 knockdown reduces poly(GP) levels in C9orf72 ALS patient iPSCs.

Control and c9ALS patient-derived iPSCs were treated with non-targeting control siRNA or RPS25-targeting siRNA. (a) Lysates from iPSCs treated with non-targeting or RPS25-targeting siRNAs were immunoblotted for RPS25 expression. Quantification illustrates that RPS25 is reduced in RPS25-targeting siRNAs (One-way ANOVA with Tukey’s multiple comparisons, n=3 independent cell culture experiments per iPSC line and condition; ****p<0.0001; mean +/− s.e.m.). (b) Immunoassay for poly(GP) levels in c9ALS iPSCs shows reduction of poly(GP) levels in RPS25 siRNA-treated cells (two-tailed, unpaired t-test; n=3 independent cell culture experiments per iPSC line and condition; ****p<0.0001, **p=0.0039, *p=0.0161; mean +/− s.e.m.). See also Fig. S5B. (c and d) RNA FISH with probe for GGGGCC (sense) RNA was used to detect and quantify sense repeat foci, pseudocolored in red. Cell nuclei are indicated in blue (Hoechst 33258). Scale bar: 5μm. (c) Control iPSCs derived from healthy subjects. (d) c9ALS-patient derived iPSCs. (e) Quantification of normalized foci per nuclei (two-tailed, unpaired t-test; n=3 independent cell culture experiments; n.s., not significant, (c9ALS #1) p=0.7234, (c9ALS #2) p=0.0654, (c9ALS #3) p=0.8189; mean +/− s.e.m.). (f) RT-qPCR of total C9orf72 mRNA (two-tailed, unpaired t-test; n=3 independent cell culture experiments; n.s., not significant, (c9ALS #1) p=0.2509, (c9ALS #2) p=0.8068, (c9ALS #3) p=0.9912; mean +/− s.e.m.). (g) RT-qPCR of C9orf72 mRNA variants harboring the repeat expansion (two-tailed, unpaired t-test; n=3 independent cell culture experiments; n.s., not significant; (c9ALS #1) p=0.5289, (c9ALS #2) p=0.8390, (c9ALS #3) p=0.4279; mean +/− s.e.m.).

Figure 3:

Figure 3:. RPS25 knockdown reduces RAN translation products and extends lifespan in a Drosophila C9orf72 model.

(a) Immunoblot of fly heads expressing 36(GGGGCC) (36R) alone or together with RpS25 RNAi in adult neurons, showing a reduction of poly(GP) levels in 36R flies expressing RpS25 RNAi. Genotypes: UAS-36(GGGGCC) /+; elavGS, UAS-36(GGGGCC) /RpS25RNAi {KK107958}; elavGS/+. (b) Quantification of blots in (a) (two-tailed, unpaired t-test; n=5 biological replicates; **p=0.0015). (c) Survival curves of male flies expressing an inducible 36(GGGGCC) construct alone or together with RpS25 RNAi. RpS25 RNAi resulted in a lifespan increase in the 36R flies (chi-squared log-rank test; ****p<0.0001). Median lifespans: C9 36R flies, 29 days; C9 36R/RpS25-RNAi, 38 days. Genotypes and n: UAS-36(GGGGCC) /+; elavGS (n=115 flies), UAS-36(GGGGCC)/RpS25RNAi{KK107958}; elavGS/+ (n=106 flies)). In separate analyses, flies expressing RpS25 RNAi alone did not alter lifespan (chi-squared log-rank test; n=83 uninduced, n=80 RNAi induced; n.s., not significant p=0.4766). Median lifespans: RpS25-RNAi uninduced, 59 days; RpS25-RNAi induced, 61 days. Genotype: UAS-RpS25RNAi{KK107958}/+; elavGS/+). (d) Expression of RpS25 RNAi together with AUG-driven codon-optimized 36 Glycine-Arginine repeats (36GR) decreases survival of male flies (chi-squared log-rank test; ****p<0.0001). Genotypes: UAS-36GR/+; elavGS (n=226 flies), UAS-36GR/RpS25RNAi{KK107958i; elavGS/+ (n=180 flies). 36R flies are codon optimized, driven by AUG and do not undergo RAN translation. (e) Quantification of surviving induced motor neurons (iMNs) derived from a c9ALS iPSC line #4–6 and 3 control iPSC lines treated with RPS25-targeting antisense oligonucleotides (ASO1 and 2) or control ASO control. The survival of HB9-RFP+ iMNs was tracked by imaging after addition of 10μM glutamate. Treatment of RPS25 ASO1 and ASO2 significantly increased survival of 3 c9ALS iMN lines ((log-rank tests; n=3 independent iMN lines per condition per treatment; ****p<0.0001; error bars, s.e.m.). (f) Relative nuclear poly(GR) quantification 3 c9ALS iMN lines treated with control or RPS25-targeting ASOs (one-way ANOVA with Tukey’s multiple comparison; n=3 independent iMN lines per condition per treatment with 20 iMNs analyzed and averaged for each n; **p=0.0055, *p=0.0105; mean +/− s.e.m.). (g) Relative nuclear poly(PR) quantification 3 c9ALS iMN lines treated with control or RPS25-targeting ASOs (one-way ANOVA with Tukey’s multiple comparison; n=3 independent iMN lines per condition per treatment with 20 iMNs analyzed and averaged for each n; (ASO1) **p=0.0017, (ASO2) **p=0.0034; mean +/− s.e.m.). For (f) and (g), individual data per c9ALS iMN line can be found in Fig. S8 and representative immunocytochemistry can be found in Fig. S10.

Comment in

References

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