Figure 4.

Overrepresentation of distal APA isoforms in the nucleus coupled to upstream intron retention. (A) Northern blot analysis of CIAO1 APA isoforms in HEK293 subcellular fractions. Probes for constitutive UTR (left), alternative UTR (middle), and the upstream intron (right) were used to corroborate the 3′READS and RNA sequencing data. (B) RNA-seq and 3′READS tracks of subcellular fractions over the CIAO1 gene. The cell line is indicated on the right of the tracks, and positioning of Northern probes used in A are shown below. (C) RNA-seq from HEK293 cells for three separate genes, all showing distal APA isoforms restricted to the nuclear fraction alongside an upstream intron retention event. For each event, proximal and distal CPA sites are shown as confirmed by 3′READS in the lower tracks. (D) RT-PCR confirming distribution of the two APA isoforms of FBXO22 in HEK293 cells as well as the presence of the retained terminal intron in the nuclear poly(A)⁺ fraction.