Fig. 6: The NXF1-NXT1 pathway regulates the circular intronic repeat RNA export.
From: Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD
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 are mean ± SEM. from three biological replicates. The exact P values for frame-GA from left to right are 0.0001, 0.0012, 0.0036, 0.0001, 0.00002, 0.0003. The exact P values for frame-GP from left to right are 0.009, 0.0093, 0.033, 0.0046, 0.0085, 0.0417. d C9ORF72-ALS iPSNs were transfected with NXT1, NXF1 or nontargeting siRNA at day 16 post-differentiation. Poly-GP was measured by ELISA after another 16 days of differentiation and maturation. Different shapes of datapoints represent independent cell lines. Data are presented as mean ± SEM. from three control and three C9ORF72-ALS cell lines. *P < 0.05 by One-way ANOVA followed by Dunn’s post hoc. e Working model: G-rich repeats stabilize introns in circular form and mediate the nuclear export. In C9ORF72-ALS/FTD, the circular intron with GGGGCCexp is the template for RAN translation in the cytoplasm, which can be upregulated by stress stimuli. The NXF1-NXT1 pathway plays an important role in the nuclear export of the circular intron, mediated by the GGGGCCexp. Source data are provided as a Source Data File.
