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. 2014 Sep 5;345(6201):1139-45.
doi: 10.1126/science.1254917. Epub 2014 Jul 31.

Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells

Ilmin Kwon  1 , Siheng Xiang  1 , Masato Kato  1 , Leeju Wu  1 , Pano Theodoropoulos  1 , Tao Wang  2 , Jiwoong Kim  2 , Jonghyun Yun  2 , Yang Xie  2 , Steven L McKnight  3
Affiliations

Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells

Ilmin Kwon et al. Science. .

Abstract

Many RNA regulatory proteins controlling pre-messenger RNA splicing contain serine:arginine (SR) repeats. Here, we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9orf72 gene altered in neurodegenerative disease encode GRn and PRn repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.

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Figures

Fig. 1
Fig. 1. CLK1/2-mediated release of GFP-fused SR domain from mCherry:hnRNPA2 hydrogel droplets
Hydrogel droplets composed of mCherry fused to the low complexity domain of hnRNPA2 were incubated with protein solution of GFP-fused to SR domains from either SRSF2 (A) or SRSF2G1/G2 (B). Both GFP proteins bound well to the mCherry:hnRNPA2 hydrogels as revealed by GFP signal trapped at the periphery of hydrogel droplets (22). Upon overnight incubation with either CLK1 or CLK2, pre-bound GFP-fused SR domain of SRSF2 was released from the mCherry:hnRNPA2 hydrogels in the presence of ATP [third and fifth panels of (A)]. The GFP-fused to the SR domain of SRSF2G1/G2 was resistant to CLK1/2-mediated release from hydrogels [third and fifth panels of (B)].
Fig. 2
Fig. 2. Native or S-to-G mutated variants of SRSF2 localize to different nuclear puncta
GFP fusion proteins linked to either the native, full-length SRSF2 or the SRSF2G1, SRSF2G2 or SRSF2G1/G2 mutants were transfected in U2OS cells in the absence (A) or presence (B) of a co-expressed mCherry:CLK1 fusion protein. The native SRSF2 protein localized to nuclear speckles and was dispersed into the nucleoplasm in the presence of co-transfected mCherry:CLK1. The SRSF2G1 and SRSF2G2 mutants localized to nucleoli as deduced by co-staining with antibodies specific to the nucleolar marker, Fibrillian. The SRSF2G1 mutant was partially redistributed from nucleoli to the cytoplasm in the presence of mCherry:CLK1. The SRSFG2 mutant was partially redistributed from nucleoli to the nucleoplasm in the presence of mCherry:CLK1. Co-expression of mCherry:CLK1 had no effect on the nucleolar localization of the SRSF2G1/G2 mutant.
Fig. 3
Fig. 3. Binding of translation products of C9ORF72 hexanucleotide repeat expansion to mCherry:hnRNPA2 hydrogel droplets
Recombinant fusion proteins linking GFP to 20 repeats of the SR, GR or PR polymers (GFP:SR20, GFP:GR20 or GFP:PR20) were applied to slide chambers containing mCherry:hnRNPA2 hydrogel droplets. After overnight incubation at 4°C, all three proteins were trapped to the periphery of the hydrogels droplets (top panels). When incubated with reaction mixtures containing either the CLK1 or CLK2 protein kinase enzymes, pre-bound GFP:SR20 was released from the hydrogels in an ATP-dependent manner. GFP:GR20 or GFP:PR20 pre-bound to mCherry:hnRNPA2 hydrogel droplets were immune to the release by CLK1 or CLK2, even in the presence of ATP.
Fig. 4
Fig. 4. Synthetic GR20 and PR20 peptides bind nucleoli and kill cells
(A) Peptides containing 20 repeats of GR or PR (GR20 or PR20, respectively) were synthesized to contain an HA epitope tag and applied to cultured U2OS cancer cells (left panels) or human astrocytes (right panels). Cells were fixed and stained with either the HA reacting antibody (green signal) or an antibody to the nucleolar protein Fibrillarin (red signal). Both GR20 and PR20 synthetic peptides associated prominently with nucleoli. Measurements of U2OS cell viability revealed toxicity in response to both PR20 (B) and GR20 (C) synthetic peptides. Cell viability was measured at 72 or 12 hours after initial treatment of PR20 or GR20, respectively. In the case of GR20 peptide, the medium was replaced every 2 hours to supplement fresh peptide. The PR20 and GR20 synthetic peptides killed U2OS cells with IC50 levels of 5.9 and 8.4 μM, respectively.
Fig. 5
Fig. 5. Effect of PR20 peptide on RNA processing
Aberrant splicing of the RAN GTPase, PTX3, NACA and GADD45A transcripts in PR20-treated cells was validated by RT-PCR (A to D). Schematic diagrams show either normal splicing (black lines) or mis-splicing (red lines). A bold red line in panel (D) indicates retention of intron. (A) RT-PCR analysis of RAN GTPase transcript: arrow indicates normal transcript (252 bp) and arrowhead indicates exon 2-skipped transcript (212 bp). (B) RT-PCR analysis of PTX3 transcript: arrow indicates normal transcript (844 bp) and arrowhead indicates exon 2-skipped transcript (442 bp). (C) RT-PCR analysis of NACA transcript: arrow indicates normal transcript (386 bp) and arrowhead indicates transcript with aberrant 5′ UTR (314 bp). (D) RT-PCR analysis of GDD45A transcript: arrow indicates normal transcript (573 bp) and arrowhead indicates intron-retention transcript (1,283 bp). (E) Scatter plot of RNA abundance measured from RNA-seq data (top panel). X-axis designates RNA abundance (log2(FPKM)) for the control sample, and the Y-axis corresponds to RNA abundance for the PR20 treated sample. Each dot represents a single mRNA species, with green dots representing transcripts of individual ribosomal protein genes. The distribution of RNA abundance fold-change between the PR20 treated sample and the control sample is shown in the bottom panel of E. Black line represents the distribution of all genes, and green line represents the distribution of ribosomal protein genes. Expression of members of the ribosomal protein gene family was significantly up-regulated by PR20 treatment (P < 2.2e-16, Kolmogorov–Smirnov test). (F) Aberrant ribosomal RNA (rRNA) processing in PR20-treated cells as analyzed by qPCR. Data are plotted as normalized fold-change against control and error is represented by standard deviation of triplicate experiments. The Y-axis indicates fold changes relative to untreated control. Black bars on X-axis below histograms indicate approximate locations of qPCR primers for 45S, 18S-5′ junction, 18S, 18S-3′ junction, 5.8S-5′ junction, 5.8S, 5.8S-3′ junction, 28S-5′ junction, and 28S rRNA (from left to right).
Fig. 6
Fig. 6. Altered EAAT2 splicing in human astrocytes exposed to PR20 synthetic peptide
(A) Normal and aberrantly spliced EAAT2 transcripts reveal locations of exon 9-skipping and intron 7 retention. Arrows indicate the primers used for RT-PCR of EAAT2 transcripts (22). (B) RNA prepared from human astrocytes exposed to 0, 10 μM or 15 μM of the synthetic PR20 peptide was interrogated with PCR primers diagnostic of the normal EAAT2 transcript (arrow), the exon 9-skipped variant (black arrowhead) or the intron 7 retention variant (grey arrowhead). No evidence of aberrant EAAT2 transcripts was observed in control cells. Cells exposed for 36 hours to 10 μM of the synthetic PR20 peptide showed equal amounts of the exon 9-skipped and intron 7 retention aberrant transcripts. Cells exposed for 36 hours to 15 μM of the synthetic PR20 peptide showed a significant increase in the amount of the exon 9-skipped EAAT2 transcript. (C) Southern blot probes specific to the exon 9-skipped and intron 7 retention aberrant EAAT2 transcripts revealed exclusive labeling of the PCR products specific to each mRNA isoform. Human astrocytes were exposed to zero, 3 μM, 10 μM or 30 μM levels of the synthetic PR20 peptide for 6 hours. Following PCR amplification and gel electrophoresis, PCR products were blotted onto nitrocellulose and hybridized with isoform-specific probes (22).
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