Fig. 5

Antisense transcription to CBF1 results in stalling mRNA transcription at the 3′-end of CBF1. a Graphical representation of the sense and antisense qPCR probes used in c and d. RT was done with an oligo-linked primer to ensure strand-specificity. qChIP probes used in e–g are indicated with a dashed black line. b Graphical visualization of the RNAPII-IP to isolate nascent RNA. c–d) RT-qPCR with the sense probes (1–3, c) and with the antisense probes (4–6, d). All probes are shown in a. Nascent RNA was compared to a total RNA preparation (steady-state level of RNA). Bars represent mean (white: 4 h 4 °C, black: 8 h 4 °C, ±SEM) from three biological replicates (rings). The relative level of transcripts was normalized to the level at 4 h 4 °C. Statistically significant differences between 4 and 8 h of 4 °C were determined with Student’s t-test (*p < 0.05, ***p < 0.001). Source data are provided as a Source Data file. e–g qChIP of total RNAPII along the CBF1 genomic region following cold exposure in WT (e), of the SVK genomic region in WT (f) and of the CBF1 transcription unit in WT and the uns-1 mutant following 4 and 8 h of 4 °C (g). The location of promoter (pCBF1), CBF1 exon, the promoter (pSVK) and SVK probes can be seen in a. Bars represent the mean of three biological replicates (grey: 0 h 4 °C, white: 4 h 4 °C, black: 8 h 4 °C, ±SEM) and are normalized to the level at 0 h 4 °C. Statistically significant differences were determined with Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). Source data are provided as a Source Data file. h Mechanistic model of how transcription of SVK represses sense CBF1 transcription. Initially during cold exposure, SVK is not expressed and sense CBF1 can be transcribed without hindrance (left panel). CBF1 expression peaks after 4 h of cold exposure. Simultaneously, expression of the lncRNA SVK is increased in the antisense direction of CBF1 (right panel). Read-through transcription of SVK results in transcription antisense to the 3′-end of CBF1 and an increase of RNAPII occupancy on both strands. The intensification of transcribing polymerases in both directions creates RNAPII collision and stalling of CBF1 sense transcription. The outcome of the collision is that fewer RNAPII complexes reach the end of the CBF1 sense transcription unit and a decrease of full-length CBF1 mRNA