Systematic generation and imaging of tandem repeats reveal base-pairing properties that promote RNA aggregation

Figures

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  Figure 1. Generation of long tandem repeats by RepEx-PCR (A) Schematic illustration of RepEx-PCR. (B) Length distribution of RepEx-PCR products. (C) Sanger sequencing results of a ∼100x CAG construct, showing the junction between 5ʹ flanking region and repeat insert (top) and a magnified view of CAG repeats (bottom). (D) Schematic illustration of a dual-luciferase reporter for detecting frameshifted CAG repeats. (E) Relative RLuc/FLuc activities of out-of-frame 0x or 100x CAG constructs. Data are shown as mean ± SD, #p > 0.1; ∗p < 0.05, two-tailed Student’s t test. (F) Percentage of clones with CAG or GGGGCC repeats inserted in the sense and antisense direction. (G) Restriction enzyme digestion of clones containing antisense (GGCCCC) repeats inserted in the original or ori-inverted vector.

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  Figure 2. Sequence determinants of CAG repeat RNA aggregation (A) Schematic illustration of the 12x MS2-tagged doxycycline-inducible repeat RNA imaging construct. (B) Representative images of cells expressing 20x (left) and ∼200x (right) CAG repeat RNAs. Scale bar, 10 μm. (C) Potential base-pairing patterns (top) and representative images (bottom) of CAG sequence variants. Substituted nucleotides are highlighted in orange. See Figure 3C for quantification. Scale bar, 10 μm.

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  Figure 3. Expanded analysis of trinucleotide repeat RNA aggregation (A) List of all of 20 non-redundant triplet repeats. Homopolymers are indicated in gray. (B) Representative images of cells expressing each of 15 triplet repeats not included in Figure 2. Scale bar, 10 μm. (C) Relationship between predicted base pairs and foci forming ability. p value, two-tailed Mann-Whitney U test.

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  Figure 4. Base-pairing properties in repeat RNA aggregation (A) Potential base-pairing pattern of ACAG repeats. The added A nucleotides are highlighted in orange. (B) Representative image (left) and quantification (right) of cells expressing 150x ACAG repeat RNA. Scale bar, 10 μm. Quantification data are shown as mean ± SD, ∗p < 0.05, two-tailed Student’s t test. (C) Predicted G-quadruplex structure of GGGAA repeats. (D) Representative image (left) and quantification (right) of cells expressing 120x GGGAA or 200x CAG repeat RNAs, treated with DMSO or PDS. Scale bar, 10 μm. Quantification data are shown as mean ± SD, #p > 0.1; ∗p < 0.05, two-tailed Student’s t test.

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  Figure 5. Aggregation of ALS/FTD-associated GGGGCC repeat RNAs (A) Potential base-pairing patterns of GGGGCC repeats. (B) Representative images of cells expressing 10x (left) or 90x (right) GGGGCC repeat RNAs. Scale bar, 10 μm. (C) Potential base-pairing patterns (top) and representative images (bottom) of cells expressing each GGGGCC variant. Substituted nucleotides are highlighted in orange. Scale bar, 10 μm. (D) Quantification of foci + cells expressing each GGGGCC variant. Quantification data are shown as mean ± SD, #p > 0.1; ∗p < 0.05, two-tailed Student’s t test.