Localization of retained introns in mouse ES cell nuclei. (A) UCSC genome browser tracks show Noc2l poly(A)+ RNA from the chromatin, nucleoplasmic, and cytoplasmic fractions. (Top) The genomic structure of the Noc2l gene is diagrammed with exon and intron probe locations in magenta, blue, and green. (Bottom) RNA-seq reads from the three fractions are displayed. Introns probed by FISH are indicated in gray. (B) FISH images of mES cells probed for Noc2l intron 12 (ATTO647N, retained), intron 4 (ATTO647N, unretained), and exons (ATTO565). Nuclear borders are outlined in white dashed lines. (C) Quantification of spot counts for the Noc2l exon and intron probes in the nuclear and cytoplasmic compartments. (D) Percentage intron retention (PIR) for Noc2l introns 12 and 4 in nuclear and cytoplasmic compartments as determined by the ratio of intron/exon costaining spots to the total exon spots in mES cells. (E) UCSC genome browser tracks show Gabbr1 poly(A)+ RNA from the chromatin, nucleoplasmic, and cytoplasmic fractions. (Top) The genomic structure of the Gabbr1 gene is diagrammed with exon and intron probe locations in green, blue, and magenta. (Bottom) RNA-seq reads from the three fractions are displayed. Introns probed by FISH are indicated in gray. (F) FISH images of mES cells probed for Gabbr1 introns 5 (ATTO647N, retained) and 11 (ATTO647N, unretained) in green and for the Gabbr1 exons (ATTO565) in magenta. Nuclear borders are outlined in white dashed lines. (G) Quantification of spot counts for the Gabbr1 exon and intron probes in the nuclear and cytoplasmic compartments. (H) PIR for Gabbr1 introns 5 and 11 in nuclear and cytoplasmic compartments as determined by the ratio of intron/exon costained spots to total exon spots in mES cells. Scale bar, 5 μm.
