Table 1.
Sequences of primers used for PCR, ChIP-qPCR, and qRT-PCR.
Fig 1.
Isoginkgetin and Madrasin are poor splicing inhibitors.
(A) Chemical structure of Madrasin and Isoginkgetin. (B) RT-PCR with primers amplifying the intron located between exons 2 and 3 of DNAJB1 or exons 4 and 5 of BRD2. HeLa cells were treated with DMSO, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h. The location of the spliced and unspliced RNA is shown on the left of the panel. The percentages of unspliced RNA compared to total (spliced + unspliced) are shown below. (C) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO, 100 μM DRB, a CDK9 inhibitor, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h. EU (green), DAPI (blue), scale bars: 50 μm. (D) Quantification of 5’EU intensity per nucleus for DMSO, DRB, PlaB, HB, Isoginkgetin, and Madrasin. Boxplot settings are: min to max values with the box showing 25–75 percentile range. > 10,000 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: n.s. not significant, **** < 0.0001. (E) Western blots of total pol II, SF3B1, α-tubulin (loading control), and histone H3 (loading control) from chromatin, nucleoplasm, and cytoplasm fractions of HeLa cells treated with DMSO, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h.
Fig 2.
Madrasin decreases transcription of protein-coding genes.
(A) Schematic of the mNET-seq technique. (B) Metagene profile of total pol II mNET-seq treated with DMSO (blue) or 90 μM Madrasin (red) for 30 min on scaled expressed protein-coding genes. (C) qRT-PCR with primers amplifying different protein-coding genes. HeLa cells were treated with DMSO or 90 μM Madrasin for 30 min (red) or 60 min (orange). cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001. (D) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks on the protein-coding gene KPNB1. The arrow indicates the sense of transcription. (E) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR across the protein-coding gene KPNB1 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001. (F) Ratios of SPT5 / total pol II, CDC73 / total pol II, or CPSF73 / total pol II from ChIP-qPCR on the intron-containing gene KPNB1 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.
Fig 3.
Transcription of intronless and histone genes is also affected by Madrasin.
(A) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red) on scaled expressed intronless genes. (B) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the intronless gene JUN. The arrow indicates the sense of transcription. (C) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR on the intronless gene JUN in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (D) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red) on scaled expressed histone genes. (E) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the histone gene H1-2. The arrow indicates the sense of transcription. (F) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR on the histone gene H1-2 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05.
Fig 4.
Isoginkgetin is a poor splicing inhibitor.
(A) RT-PCR with primers amplifying the intron located between exons 2 and 3 of DNAJB1 or the intron located between exons 4 and 5 of BRD2. HeLa cells were treated with DMSO, 30 μM Isoginkgetin for 1h to 6h, or 1 μM PlaB for 6h. The locations of the spliced and unspliced RNA are shown on the right of the panel. The percentages of unspliced RNA compared to total (spliced + unspliced) are shown below. (B) Percentage of spliced reads in total RNA-seq treated with DMSO (blue) or Isoginkgetin (red) for the time indicated on the figure. Each point represents a biological replicate. Statistical test: Wilcoxon rank sum test. P-value: n.s. not significant, * < 0.05, **** < 0.0001. (C) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. EU (green), DAPI (blue), scale bars: 50 μm. (D) Quantification of 5’EU intensity per nucleus for DMSO and Isoginkgetin. Boxplot settings are: min to max values with the box showing 25–75 percentile range. 8,466 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001. (E) qRT-PCR with primers amplifying a region from the pol I transcribed pre-rRNA. HeLa cells were treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: n* < 0.05.
Fig 5.
Isoginkgetin treatment affects pol I and pol II transcription.
(A) qRT-PCR with primers amplifying different protein-coding genes. HeLa cells were treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. cDNA was generated with oligo(dT). Values are normalised to the protein-coding gene GAPDH and shown as relative to DMSO, mean ± SEM, n = 4 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (B) Total pol II ChIP-qPCR on the intron-containing gene KPNB1 in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01. (C) Total pol II ChIP-qPCR on the intronless gene JUN in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05. (D) Total pol II ChIP-qPCR of the histone gene H1-2 in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05. (E) Western blots of total pol II, Ser2-P, Ser5-P, and histone H3 on the chromatin fraction of HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h, 2h, or 4h. Quantifications of the western blots are shown below each panel. Each quantification has been normalised to histone H3 signal and then to the DMSO condition. (F) ChIP-qPCR of total pol II, Ser2-P, and Ser5-P across the intron-containing gene KPNB1 in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h, 1h30, or 2h. Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.