Figure 6.

A subset of splice sites is affected by inhibiting CDK12^AS for a short time.

Bru-Seq and BruChase-Seq were carried out as described in the text. An internal section of the AMOTL1 gene is shown. The top set of 4 traces (Bru-Seq) show the relative transcription rate (incorporation of BrU in 30 min pulse) in regular HeLa cells (WT) and in CDK12^AS HeLa cells in the absence or presence of inhibitory analog 1-NM-PP1. The gold trace (CDK12^AS cells + analog) begins to drop below the other three downstream of exon 6 (e6). The bottom set of 6 traces (BruChase-Seq) shows that in ‘WT cells in the presence of analog (blue, green, black), after a 30 min pulse of BrU and a 2 hour chase, exon signals predominate (four exons are indicated by vertical pink dotted lines). The introns are spliced out and degraded (valleys between WT peaks). In contrast, in CDK12^AS cells treated identically, the intron following exon 3 (e3) is not spliced out and is “retained.” But the next intron is spliced out normally, and then intronic sequences past exon 6 (e6) are retained. Apparently only part of the intron is retained, since the pulse-chase (BruChase-Seq) traces return to normal about a third of the way through the intron. Farther downstream, splicing looks ~ normal.