RNA phase transitions in repeat expansion disorders (original) (raw)

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Acknowledgements

We thank M. K. Rosen, W. W. Seeley, and the members of the Vale laboratory for discussions. A.J. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2181-14).

Author information

Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California, San Francisco, 94158, California, USA
    Ankur Jain & Ronald D. Vale
  2. Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, 02543, Massachusetts, USA
    Ankur Jain & Ronald D. Vale

Authors

  1. Ankur Jain
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  2. Ronald D. Vale
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Contributions

A.J. and R.V. designed research and wrote the paper. A.J. performed experiments and analysed data.

Corresponding author

Correspondence toRonald D. Vale.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks C. P. Brangwynne, M. Carmo-Fonseca, J. Shorter and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Disease-associated repeat-containing RNAs form clusters in vitro.

a, Fluorescence micrographs for RNA with various GC content compared against RNA with disease-associated repeat expansions. Sequences of the DNA templates used for transcription are provided in Supplementary Table 2. b, Fluorescence micrographs comparing 47×CUG RNA and a corresponding control RNA (Scr1) with identical base composition but with a scrambled sequence. Similarly, 47×CAG RNA was compared with two different RNAs (Scr2, Scr3) having the same base composition as 47×CAG but whose sequences were scrambled. The extent of inhomogeneity was quantified by the index of dispersion (σ_2/μ) across more than 20 independent imaging areas (1,800 μm2 each). Each data point represents an independent imaging area. c, Representative micrographs of 47×CAG RNA clusters at indicated concentrations. Spherical RNA clusters are observable up to 25 nM RNA concentration. In this concentration regime, the reaction is reactant limited and the cluster size is below the diffraction limit. RNA clustering at all concentrations was not observed in the presence of 100 mM ammonium acetate. Representative images at the indicated RNA concentration in the presence of 100 mM NH4OAc (+ NH4OAc). d, RNA enrichment in the clusters. Left, Cy3-labelled 66×CAG RNA was serially diluted in conditions preventing RNA clustering (10 mM Tris pH 7.0, 10 mM MgCl2, 100 mM NaCl), and the bulk solution fluorescence was calibrated against the RNA concentration. The enrichment of RNA in the clusters was determined by comparing their fluorescence intensity against this calibration. When the input RNA concentration was 100 ng μl−1, the concentration in the clusters corresponded to ~16.3 μg μl−1, or an enrichment of 163-fold. a.u., arbitrary units. Right, RNA clusters were precipitated by centrifugation at 16,000_g for 10 min at room temperature. The concentration of the soluble RNA after centrifugation was determined by measuring absorbance at 260 nm. The concentration of the RNA in the solution phase decreased with the increasing CAG-repeat number. e, f, The 47×CAG RNA clusters were treated with proteinase K (60 U ml−1), DNase I (200 U ml−1), or RNaseA (0.7 U ml−1) for 10 min at room temperature. Representative micrographs (e) and quantification (f). g, Clustering of 47×CAG RNA is inhibited by NaCl. h, Binary phase diagram for 1.25 μM 47×CAG RNA as a function of MgCl2 and NaCl concentrations. Blue dots represent two-phase regime while the red dots indicate a homogenous single-phase regime. i, The RNA clusters were in a solid-like state and did not exhibit fluorescence recovery upon photobleaching, as indicated by the representative micrographs for 47×CUG (top) and 47×CAG (bottom) RNA at the indicated time points. j, Sample images showing aborted fusion events between 47×CAG RNA clusters, suggesting that the clusters were liquid-like initially and later underwent a liquid-to-solid transition. Fusion events were probably aborted as the clusters solidified before relaxation to a spherical geometry. Sites of aborted fusion are marked by arrows. Scale bars in a–c, e, g, 5 μm, in i, j, 1 μm. Data are median ± interquartile range. Data are representative of at least three independent experiments across at least two independent RNA preparations.

Extended Data Figure 2 Base-pairing interactions impart solid-like properties to DNA–spermine complexes.

Electrostatic interactions between polymeric anions (such as nucleic acids) and multivalent cations can lead to phase separation via formation of polyelectrolyte complexes, a phenomenon known as complex coacervation. We found that spermine, a tetravalent cation at pH 7, could induce phase separation of single-stranded DNA oligonucleotides. a, Mixing 10 mM spermine pH 7 (left tube) with 10 μM T-90 DNA (90-mer polyT DNA oligonucleotide) (right tube) immediately resulted in a turbid solution (centre tube). b, Examination by bright-field microscopy revealed numerous spherical droplets. Using a fluorescently labelled T-90, we confirmed that the droplet phase was enriched in DNA. Representative bright-field (left), fluorescence (centre), and overlay (right) images for the DNA–spermine complexes. c–f, We investigated the effect of base-pairing interactions on DNA–spermine complexes. The T-90 DNA–spermine complexes were liquid-like, as evidenced by their spherical geometry (c) and a rapid FRAP (f, 99 ± 1% recovery, _τ_FRAP-T90 = 5 ± 2 s, mean ± s.d., n = 3 droplets). Next, we designed a 90-base-long DNA with five 8-bp palindromic hybridization sites separated by poly-dT spacers (sequence S1, sequences in Supplementary Table 2). S1 DNA also phase-separated and formed spherical liquid-like droplets in the presence of spermine (d). However, the S1-spermine droplets exhibited reduced fluidity, as evidenced by a slower recovery upon photobleaching (f, 90 ± 4% recovery, _τ_FRAP-S1 = 335 ± 41 s, mean ± s.d., n = 5). e, We performed similar spermine-mediated coacervation experiments with a (dAdT)45 oligonucleotide (AT-45) that could form multivalent A:T base-pairing interactions. AT-45 DNA formed interconnected network-like structures spanning hundreds of micrometres or gels (e). These AT-45 DNA gels were in a solid-like state, as evidenced by lack of FRAP (f, 14 ± 5% recovery, mean ± s.d., n = 4 clusters). Scale bars, 5 μm.

Extended Data Figure 3 Disease-associated, repeat-containing RNAs coalesce into nuclear foci in cells.

a, b, Expression of 47×CAG (a) and 120×CAG (b) RNA leads to the formation of nuclear puncta. Representative images (left) and quantification of the percentage of cells showing RNA foci (right) with and without induction; n, number of cells analysed. c, Time-lapse images of 120×CAG RNA accumulation in the nuclei of U-2OS cells. Cells were induced with 1 μg ml−1 of doxycycline at t = 0. See also Supplementary Video 3. d, Number of foci per cell increased with increasing 47×CAG RNA expression levels. The expression levels were controlled by increasing the virus titre. e, The 47×CAG RNA accumulated in the nuclei as puncta, while control RNA with coding (mCherry) or a non-coding sequence (mCherry′, reverse complement of mCherry sequence) did not form nuclear inclusions, as shown in the representative MS2–YFP fluorescence micrographs (left) and quantification of the number of foci per cell (right). f, U-2OS cells were transduced with the indicated constructs tagged with 12×MS2 hairpins under a tetracycline-inducible promoter. RNA was visualized by FISH using Cy3-labelled oligonucleotide probes against MS2-hairpins. Representative micrographs showing the localization of mCherry (top), 47×CAG (middle), and 29×GGGGCC (bottom) RNA with (+ Tet) or without (− Tet) doxycycline induction. The probes did not bind in the absence of induction. Nuclei are counterstained with DAPI (depicted in blue). g, Intensity distribution for single RNA spots in cells expressing 5×CAG (top) and in the cytoplasm of cells expressing 29×GGGGCC (bottom). h, RNA copy number was determined by dividing the total Cy3 fluorescence intensity in a cell by that of a single RNA, as determined in g. The 47×CAG RNA copy number corresponds to the highest viral titre used in d. Similar results were obtained using NanoString (8,800 ± 1,500 copies per cell for 47×CAG RNA, mean ± s.d., n = 3 independent experiments). i, Induction of 47×CAG RNA foci did not cause overt toxicity or a reduction in cell division rates over 7 days. Normalized cell counts in 47×CAG-transduced cells with or without doxycycline induction. Cell counts were normalized to control cells (without 47×CAG transduction), grown under corresponding induction conditions. Each data point in d, e, h represents one cell, and data are shown as median ± interquartile range. Data points in i represent technical replicates, and are shown as mean ± s.d. Scale bars, 5 μm.

Extended Data Figure 4 Identification of RNA foci in live cells.

a–c, We used a fluorescence-intensity and size-based threshold to identify RNA foci. In brief, U-2OS cells expressing the RNA of interest together with MS2CP–YFP were imaged using a spinning disk confocal microscope, and 0.3 μm _Z_-stacks were acquired (a). To account for variability in MS2CP–YFP expression levels, we used a cell-intrinsic intensity threshold for foci identification. We manually segmented the nuclei (b) and determined the mean fluorescence intensity in the nucleus. RNA foci were identified using the FIJI 3D Objects Counter plugin, with an intensity threshold as 1.6× the mean fluorescence intensity in the nucleus of the cell, and a size cut-off of more than 50 adjoining pixels (pixel size, 83 nm × 83 nm). This algorithm accurately identified the foci as depicted in c. d–h, We compared the extent of foci formation in 47×CAG and 5×CAG expressing cells. d, The mean nuclear fluorescence intensity was similar between the 47×CAG and 5×CAG expressing cells. The cells were compared via various metrics: e, number of foci per cell; f, total volume of foci per cell; g, integrated fluorescence intensity of the foci per cell; and h, normalized variance in the fluorescence intensity in the nucleus per cell. Scale bar, 5 μm. Data are median ± interquartile range.

Extended Data Figure 5 CAG RNA foci co-localize with nuclear speckles.

Representative immunofluorescence micrographs depicting that the 47×CAG RNA foci co-localized with the marker for nuclear speckles (SC-35) but not with other nuclear bodies such as PML bodies (PML), paraspeckles (nmt55), nucleoli (Fib1), or Cajal bodies (coilin). RNA foci were stained using an antibody against GFP. Nuclei were stained with DAPI. Data are representative of three or more independent experiments. Scale bars, 5 μm.

Extended Data Figure 6 RNA foci are disrupted by treatments that prevent RNA gelation in vitro.

a, RNA FISH using a probe directed against MS2 hairpin loops confirmed that 47×CAG RNA foci were disrupted by treatment with 100 mM NH4OAc, thus precluding the possibility that the observed disruption of RNA foci in live cells was due to dissociation of MS2CP–YFP from the MS2 hairpins. Representative images and corresponding quantification. b, Transfection of an 8×CTG oligonucleotide disrupted 47×CAG RNA foci while control oligonucleotides (3×C4G2 or Control) did not. Representative images and quantification of the number of RNA foci per cell. Sequences of the oligonucleotides are provided in Supplementary Table 2. c, Doxorubicin (Dox) disrupted 47×CAG RNA clustering in vitro in a dose-dependent manner. Representative micrographs and the quantification of the inhomogeneity in the solution at indicated RNA and doxorubicin concentrations. d, RNA FISH using a probe directed against MS2 hairpin loops confirmed that 47×CAG RNA foci were disrupted by treatment with 2.5 μM doxorubicin, suggesting that the observed disruption of RNA foci in live cells was probably not an artefact of MS2CP–YFP dissociation from MS2 hairpins. Scale bars, 5 μm. Data are median ± interquartile range. Data are representative of three or more independent experiments.

Extended Data Figure 7 Doxorubicin disrupts RNA foci but not nuclear speckles.

a, Representative immunofluorescence micrographs of U-2OS cells expressing 47×CAG stained with antibodies against GFP (MS2–YFP) and SC-35, as a marker for nuclear speckles. b, c, Treatment with 2 μM tautomycin for 4 h (b) or 100 mM NH4OAc for 10 min (c) disrupted both the 47×CAG RNA foci as well as the nuclear speckles. d, Treatment with 2.5 μM doxorubicin for 2 h specifically abrogated the 47×CAG RNA foci but the nuclear speckles were not disrupted. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm. e, Quantification of the total volume occupied by nuclear speckles (left) and the integrated intensity of the SC-35 immunofluorescence (right) per cell under various treatments. Data are median ± interquartile range. Data are representative of three or more independent experiments.

Extended Data Figure 8 Doxorubicin disrupts RNA foci in fibroblasts from patients with DM1.

a, Fibroblasts derived from patients with DM1 (DM1a, DM1b) or control fibroblasts (Hs27) were stained using a FISH probe directed against expanded CUG repeats (8×CAG labelled with Atto647N). Representative fluorescence images showing RNA foci in DM1 cell lines but not in control. Nuclei were counterstained with DAPI (blue). b, Quantification of the number of RNA foci per cell across various cell lines. c, Single-molecule FISH using probes designed against the wild-type DMPK allele showed isolated diffraction-limited spots in control fibroblasts (Hs27), indicated by white arrows, probably arising because of single mRNA. In the patient-derived fibroblasts (DM1a, DM1b), isolated spots (white arrows) as well as several bright puncta (yellow arrows) were observed. Since both the wild-type and the mutant transcript with expanded CUG repeats could each accommodate the same number of fluorescent probes (48 probes per transcript), the higher brightness indicates that each punctum in cells derived from patients with DM1 (yellow arrows) contained multiple DMPK mRNAs. d, Treatment with 2 μM doxorubicin for 24 h reduced the average number of RNA foci per cell by 66% and the total volume of foci per cell by 87%. Representative images with or without doxorubicin treatment and corresponding quantification. Data are aggregated from two independent experiments, and are representative of four or more independent experiments. Scale bars, 5 μm. Data are median ± interquartile range.

Extended Data Figure 9 GGGGCC repeat-containing RNAs form clusters in vitro and foci in cells.

a, Binary phase diagram for 23×GGGGCC RNA clustering in vitro as a function of NaCl and MgCl2 concentrations. RNA concentration was 1.5 μM. Blue dots represent two-phase regime while the red dots indicate a homogenous single-phase regime. b, Representative fluorescence micrographs for 3×GGGGCC RNA clusters before and after photobleaching at indicated time points. The lack of fluorescence recovery indicated that the RNA in the clusters was immobile or in a solid-like state. Scale bar, 1 μm. c, Quantification of the number of RNA foci per cell for U-2OS cells expressing 29×GGGGCC or 29×CCCCGG RNA. d, Number of 29×GGGGCC RNA foci increased with the increasing level of RNA expression. The expression levels were controlled by increasing the virus titre. e, Representative fluorescence micrographs and corresponding quantification of the total volume of foci per cell in U-2OS cells transduced with 12×MS2 tagged RNA with the indicated number of GGGGCC repeats. f, GGGGCC RNA foci exhibited incomplete recovery upon fluorescence photobleaching. Representative fluorescence micrographs for 29×GGGGCC RNA foci at indicated time points. g, Fluorescence recovery plots for GGGGCC RNA foci with indicated number of repeats. Data are average of n = 10 foci at each repeat number. h, Percentage of 29×GGGGCC RNA retained in the nucleus compared against a control RNA encoding for mCherry. i, Effect of flanking sequences on the formation of GGGGCC RNA foci. Construct G1 had 29×GGGGCC repeats with 12×MS2 hairpins (~0.7 kb) downstream of the repeats for RNA visualization. Incorporation of a ~1 kb long sequence (G2, sequence in Supplementary Table 1) between the 29×GGGGCC repeats and 12×MS2 repeats did not inhibit foci formation. Similarly, RNA foci were observed in construct G3, which had the same 5′ flanking sequence as found in the endogenous locus in intron 1 of c9orf72. However, incorporation of a longer ~1 kb 5′ flanking sequence (G4) inhibited the formation of RNA foci. j, Transfection of U-2OS cells with a 3×CCCCGG ASO disrupted the 29×GGGGCC RNA foci while a control ASO did not. Representative micrographs and the quantification of the number of RNA foci per cell. k, Representative fluorescence micrographs and corresponding quantification of inhomogeneity for 23×GGGGCC RNA in vitro with or without 1 mM doxorubicin. l, Same as k with or without 100 mM NH4OAc. Scale bars in e, f, j–l, 5 μm. Data are shown as median ± interquartile range (c–e, i–l) or mean ± s.d. (g, h). Data are representative of three or more independent experiments.

Extended Data Figure 10 GGGGCC RNA foci co-localize with nuclear speckles.

Representative immunofluorescence images illustrating that the 29×GGGGCC RNA foci co-localized with the marker for nuclear speckles (SC-35) but not for Cajal bodies (coilin). The GGGGCC RNA foci also recruited endogenous hnRNP H and MBNL1. Scale bars, 5 μm. Data are representative of three or more independent experiments.

Supplementary information

Supplementary Table 1

This table contains sequences of plasmids. (XLSX 60 kb)

Supplementary Table 2

This table contains sequences of DNA oligonucleotides and transcription templates. (XLSX 42 kb)

Fluorescence recovery after photobleaching (FRAP) for RNA clusters in vitro

A section of 47xCAG RNA cluster, ~1 μm diameter, was photobleached and the fluorescence was monitored over time. The photobleached region did not exhibit appreciable recovery over ~10 min. Scale bar is 5 μm. (AVI 992 kb)

Base pairing interactions affect fluorescence recovery rates for phase separated DNA

T-90 (left), sequence S1 (center) or AT-45 (right) phase separate in the presence of spermine. ~1 μm diameter region was photobleached and the fluorescence recovery was monitored over time. The fluorescence recovery rate decreases with increasing base pairing. Scale bars represent 5 μm. (AVI 3403 kb)

Real-time visualization of RNA foci formation

U-2OS cells were transduced with a 120xCAG RNA, tagged with 12xMS2 hairpins. RNA is visualized by coexpression of MS2CP-YFP. Expression of 120xCAG RNA was induced at t = 0 min and cells were visualized using confocal microscopy. Scale bar is 5 μm. (AVI 4868 kb)

RNA foci are liquid-like and undergo fusion events

Cells were transduced to express MS2-tagged 47xCAG RNA, and RNA was visualized by co-expression of MS2CP-YFP. Two or more RNA foci in proximity coalesce in to a single punctum. A typical fusion event is marked by an arrow. Scale bar is 5 μm. (AVI 586 kb)

FRAP for 47xCAG RNA foci

47xCAG RNA punctum was photobleached at t = 0 and the fluorescence recovery was monitored over time. Arrow indicates the site of photobleaching. Scale bar is 5 μm. (AVI 1008 kb)

Partial bleaching for 47xCAG RNA foci

A region ~1 μm in diameter was photobleached in a 47xCAG RNA punctum and the fluorescence recovery was monitored over time. Arrow indicates the site of photobleaching. Scale bar is 5 μm. (AVI 1451 kb)

FRAP for 47xCAG RNA foci after ATP depletion

Cellular ATP was depleted in cells expressing 47xCAG RNA. An RNA punctum was photobleached at t = 0 and the fluorescence recovery was monitored over time. Arrow indicates the site of photobleaching. Scale bar is 5 μm. (AVI 2350 kb)

Effect of ammonium acetate on RNA foci

U-2OS cells expressing 47xCAG RNA were treated with 100 mM ammonium acetate at t = 0. RNA is visualized by coexpression of MS2CP-YFP. RNA foci disassemble within minutes after addition of ammonium acetate. Scale bar is 5 μm. (AVI 25633 kb)

FRAP for 29xGGGGCC RNA foci

29xGGGGCC RNA punctum was photobleached at t = 0 and the fluorescence recovery was monitored over time. Arrow indicates the site of photobleaching. Scale bar is 5 μm. (AVI 3174 kb)

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Jain, A., Vale, R. RNA phase transitions in repeat expansion disorders.Nature 546, 243–247 (2017). https://doi.org/10.1038/nature22386

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