Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD

Fig. 1. C9ORF72 repeat-containing introns form nuclear RNA granules and are exported into cytoplasm. a Schematic of the C9ORF72 splicing reporter construct. MBS/PBS: MS2/PP7 binding sites; ex1a, ex2: exon 1a and 2 of C9ORF72 gene. Two sets of RNA FISH probes were designed to target either 24×MBS or 24×PBS with different fluorescent dyes. The mature mRNA only shows PBS signal, spliced intron only has MBS signal, and the unspliced pre-mRNA contains both MBS and PBS signals. b Representative images of two-color smFISH experiment to study the spatial distribution of RNA species in control or +(GGGGCC)70 reporter cells. The boxes 1–4 were enlarged on the right. Magenta: intron (MBS); cyan: exon (PBS); blue: DAPI; arrow: single-intron molecule; arrowhead: single exon molecule. Scale bar: 5 μm and 1 μm for zoom in, respectively. Quantification shown in panels c–g. c Number of RNA granules per nucleus. Repeat-containing introns formed large granules colocalized with exons (gray) as well as intron only (pink) granules. Cells were treated with transcription inhibitor actinomycin D (1 μg/mL for 20 min) to exclude the effect of transcription. Data are mean ± SD from three biological replicates. The…

Fig. 2. The distribution and molecular identity of endogenous C9ORF72 RNAs in patient fibroblast cells. a Schematic of smFISH probes targeting endogenous C9ORF72 RNAs. A set of 76 probes targets intron 1 (magenta) and a 77-probe set targets all the exons (cyan). b Representative two-color smFISH images of endogenous C9ORF72 RNAs in C9ORF72-ALS patient-derived fibroblasts. arrow: single-intron molecule; arrowhead: single exon molecule. Scale bar: 5 μm, and 1 μm for zoom in. c Quantification of nuclear introns, mRNAs and pre-mRNAs per nucleus. Datapoints of different shapes represent individual fibroblast line derived from two healthy controls and three C9ORF72-ALS patients. The horizontal bar indicated the mean value. d, e The percentage of cells containing different numbers of cytosolic exons (d) and introns (e) were quantified and calculated from each line with three technical replicates. Datapoints of different shapes represent individual fibroblast line derived from two healthy controls and three C9ORF72-ALS patients. The quantified cell number: Ctrl-1 (81), Ctrl-2 (165), C9-2 (135), C9-5 (131), and C9-6 (93). f Cytoplasmic introns were not colocalized with exons in C9ORF72-ALS…

Fig. 3. The cytoplasmic repeat-containing introns are exported in the circular form. a Schematic of splicing and circular RNA enrichment by RNase R treatment. RNase R only degrades linear RNA. The tail of the lariat can be degraded, but the circular part will be preserved. b Representative images of two-color smFISH in +(GGGGCC)70 reporter cells treated with RNase R (bottom) or just buffer (top). Magenta: intron (MBS); cyan: exon (PBS); blue: DAPI; arrow: intron; arrowhead: exon. Scale bar: 5 μm, and 1 μm for zoom in. Quantification shown in panels c and d. c, d Scatter plot of intron vs exon numbers in the cytoplasm (c) or nucleus (d) of each cell without RNase R (left) or with RNase R treatment (right). Each dot represented one cell. Total three technical replicates were carried out: 287, 289, 263 cells quantified for RNase R treatment and 222, 369, 342 cells for control (without RNase R treatment). e Deep sequencing identified circular boundaries of intron RNAs. The major splicing junction in mRNAs and branch site in circular RNAs were annotated. The libraries were prepared using the indicated primers (P1–P2 for circular intron; P3–P4 for mRNA), and the whole fragments were sequenced…

Fig. 4. G-rich repeats stabilize the spliced intron and mediate the nuclear export. a Schematic of splicing reporter constructs with four other repeat sequences in the context of C9ORF72 gene. The GGGGCCexp was replaced with the indicated repeats in the reporter of Fig. 1a. MBS/PBS: MS2/PP7 binding sites; ex1a, ex2: exon 1a and 2 of C9ORF72 gene. From these reporters, the mature mRNA only shows PBS signal, spliced intron only has MBS signal, and the unspliced pre-mRNA contains both MBS and PBS signals. b, c Representative images of two-color smFISH of C9ORF72 splicing reporter cells containing intronic (CGG)98 (b) and (CCCCGG)70 (c) repeats. Scale bar: 5 μm, and 1 μm for zoom in. d, e Quantification of cytoplasmic intron (d) and exon (e) numbers per cell in the splicing reporter cell lines with four different repeat expansions. Each symbol represented a single cell and the three shapes represented three biological replicates (n = 75, 80, 73 cells in (CGG)98; n = 162, 146, 103 cells in (CCCCGG)70; n = 147, 171, 119 cells in (CCTG)240; n = 180,114,151 cells in (ATTCT)73). The mean of each replicate (larger black shapes) was used to calculate the average (horizontal bar) and SD (error bars)…

Fig. 5. Translation of circular repeat-containing intron. a Diagram of single-molecule translation imaging reporter for C9ORF72 repeat-containing intron. The SINAPS reporter lacking the ATG start codon was inserted after the (GGGGCC)70 repeats in the GR frame in the splicing construct. The SunTag fluorescence (green, scFv-sfGFP) on the RNA represents the RAN translation signal. The auxin-inducible degron (AID) at the end of the reporter was used to degrade the mature proteins and decrease the background signals21. b Live cell imaging illustrates that the fluorescence puncta are translation sites. U-2 OS cells stably expressing membrane tethered stdMCP protein31, 57 were transiently transfected with intronic translation reporter in a. Cells were treated with translation inhibitor puromycin (100 μg/mL) at time 0 and imaged at 10 min. Translation was shut down efficiently. Green (protein): SunTag-scFv-sfGFP; magenta (intron): stdMCP-Halotag-CAAX-JF646. Scale bar: 2 μm. Total three independent biological replicates were examined with similar results. c Representative smFISH & IF images to measure RNA and translation. Green: protein; magenta: intron; cyan: exon. Arrow: intron; arrowhead:…

Fig. 6. The NXF1-NXT1 pathway regulates the circular intronic repeat RNA export. a Two-color smFISH in +(GGGGCC)70 splicing reporter cells transfected with nontargeting control (top) or NXF1-targeting (bottom) siRNA. Magenta: intron (MBS); cyan: exon (PBS); blue: DAPI. Scale bar: 5 μm. The boxes were zoomed and shown on the right, scale bar: 1 μm. Arrow: intron; arrowhead: exon. Dash line: nuclear boundary. Quantification shown in panel b. b Scatter plot of cytosolic intron vs exon numbers in each cell with either nontargeting control siRNA (top) or NXF1-targeting siRNA (bottom) transfection. Each dot represented one cell (total 418 cells for control and 490 for NXF1 siRNA, from three biological replicates). c HeLa Flp-In bicistronic splicing reporter cells (C9R-NLuc is located in the C9ORF72 intron 1, flanked by C9 exon 1 and 2, pA: poly-A tail)35 were induced to express translation reporters by doxycycline after two days of siRNA transfection, and luciferase activities were measured after another 24 h. NLuc signals were normalized to FLuc in each sample and the relative expression was compared to nontargeting siRNA control. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. Data…