Journal: Nature Communications
Article Title: Nuclear export and translation of circular repeat-containing intronic RNA in C9ORF72-ALS/FTD
doi: 10.1038/s41467-021-25082-9
Figure Lengend Snippet: 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 quantified cell number: Ctrl (88, 82, 41) and (GGGGCC) 70 (72, 43, 73). d Scatter plot of intron vs exon numbers in each RNA granule. Each dot represented one granule (from three biological replicates). The lines were linear fit to the scatter plot. The slope reflects the ratio of intron vs exon molecules in each granule. e Scatter plot of the numbers of single introns vs exons in each nucleus. Each dot represented a single nucleus. The lines were linear fit to the scatter plot. The slope reflects the ratio of intron vs exon molecules in each nucleus. f , g Quantification of cytoplasmic intron ( f ) and exon ( g ) number per cell. Each symbol represented a single cell and the three shapes represented three biological replicates. The mean of each replicate (larger black shapes) was used to calculate the average (horizontal bar) and SD (error bars) in each group, as well as for statistic comparison between groups. * P < 0.05, ** P < 0.01, two-tailed t -test. The cell number in d – g : Ctrl (60, 43, 93) and (GGGGCC) 70 (93, 95, 49). h Percentage of cytoplasmic introns colocalized (unspliced) or uncolocalized (spliced) with exons in +(GGGGCC) 70 cells. The exported GGGGCC repeat-containing RNAs are predominantly spliced introns. Source data are provided as a Source Data File.
Article Snippet: To determine the coordinates and fluorescence amplitudes of smFISH and translation site signals, images were analyzed using the Matlab software FISH Quant .
Techniques: Construct, Binding Assay, Two Tailed Test
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