(
A) Violin plot showing the ratio of transcription in G1 phase versus G2 phase for the genes belonging to the
immediate decrease or
delayed decrease groups (
Figure 2A and
Supplementary file 1). Data were retrieved from
Battich et al., 2020. New transcripts were labeled for 30 min using EU, and old and new transcripts were quantified using deep sequencing. The relative rate of transcription was defined as the number of labeled transcripts in G1 versus G2 phase. Dashed line indicates supplemnetary filea ratio of 1, indicative of a similar transcription rate in G2 and G1 phase (ratios < 1 are indicative of reduced transcription in G1 phase). (
B–G) mRNA abundance over time for genes that undergo mRNA decay at the mitosis-to-G1 (M-G1) transition. Blue lines indicate the best fit obtained using the mathematical model described in
Figure 3C. Representative genes of the
immediate decrease group (CDK1, TOP2A, and UBE2C) and the
delayed decrease group (CENPA, ALR6IP1, and UBALD2) are shown. (
H) Histogram of the time (relative to metaphase) when mRNA levels start to decline is shown for genes that are downregulated during the M-G1 phase transition. mRNA levels over time were fit as in
B–G and the onset time of mRNA decline was determined for each of the 220 downregulated genes. (
I) Histogram of mRNA half-lives for the 220 genes that are downregulated during the M-G1 phase transition. To obtain mRNA half-lives, mRNA levels over time were fit as in
B–G and the mRNA half-lives were calculated using the mathematical model described in
Figure 3 (see Materials and methods). (
J–L) Comparison of mRNA half-lives during the M-G1 phase transition with mRNA half-lives in asynchronous cells determined in previous studies (
Herzog et al., 2017;
Schwanhäusser et al., 2011;
Tani and Akimitsu, 2012). Dashed lines indicate identical half-lives. Note that the half-lives of most genes are shorter during the M-G1 phase transition than in asynchronous growing cells. (
M) Boxplot of mRNA half-lives of
immediate and
delayed decrease genes. For each gene, the half-live was determined from the moment mRNA levels start to decrease (see
Supplementary file 1). (
N) Analysis of transcription inhibition by Actinomycin D. Expression levels of the DNA damage-induced gene CDKN1a were measured by RT-qPCR in cells that were DNA damaged (exposed to 5 Gy ionizing radiation), in the presence or absence of Actinomycin D, relative to non-irradiated cells. Lines with error bars indicate average ± SEM of three experiments. (
O) Mitotic index of RPE-1 cells treated with the transcription inhibitor Actinomycin D. RPE-1 cells were arrested in G2 phase using a CDK1 inhibitor (RO-3306). After 16 hr, the CDK1 inhibitor was removed and replaced by Taxol, thereby releasing cells from the G2 phase arrest and blocking cells in mitosis. Forty-five minutes later, mitotic cells were collected through mitotic shake-off, after which Actinomycin D was added for up to 2 hr. Cells were fixed and the fraction of mitotic cells was determined by fluorescence activated cell sorting (FACS) (by staining cells for DNA content and the mitosis-specific marker phosphorylated histone 3 at ser 10). Lines with error bars indicate average ± SEM of three experiments. p-Values are based on a one-tailed Student’s t-test, and indicated as * (p < 0.05), ** (p < 0.01), *** (p < 0.001), ns = not significant.