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. 2020 Nov 11;9:e59770. doi: 10.7554/eLife.59770

Figure 3. Alpha-satellite RNAs are products of Pol II-mediated transcription.

(A) Treatment of HeLa cells with small-molecule inhibitors reveals that alpha-satellite transcripts are mediated by RNA polymerase II. Cells were treated with the RNA Polymerase I inhibitor BMH-21 (24 hr), the RNA Polymerase III inhibitor ML-60218 (24 hr), or the Cdk7 inhibitor THZ1 (5 hr), which inhibits RNA Polymerase II initiation. Transcripts were identified using the ASAT smFISH probe set. (B) Quantification of smFISH foci from (A) after treatment of HeLa cells with small-molecule inhibitors against Cdk7, RNA Pol I, and RNA Pol III. smFISH foci were substantially reduced after inhibition of RNA Pol II activator, Cdk7, but increased by RNA Pol I inhibition. Error bars represent the mean and standard deviation of at least 240 cells. (C) Graph showing independent replicates of ASAT smFISH foci for each small-molecule inhibitor treatment (Cdk7, RNA Pol I, and RNA Pol III). P-values represent T-tests for the indicated comparisons. (D) RT-qPCR quantification reveals significantly reduced levels of chromosome 21 alpha-satellite transcripts of cells treated by the Cdk7 inhibitor THZ1 for 5 hr, but increased levels following RNA polymerase I inhibition (24 hr treatment) when compared to control HeLa cells. The levels of alpha-satellite RNA from chromosome 21 detected was outside of our quantifiable range in cells treated with CDK7 inhibitor and thus was set to 0. The mean of 3 biological replicates was plotted and error bars represent the standard deviation. P-value represents the results of a T-test.

Figure 3—source data 1. Source data for the RT-qPCR experiments shown in Figure 3D.

Figure 3.

Figure 3—figure supplement 1. Analysis of centromere RNAs following RNA polymerase inhibition.

Figure 3—figure supplement 1.

(A) Treatment of HeLa cells with small-molecule inhibitors against each polymerase reveals that alpha-satellite transcripts are mediated by RNA polymerase II (24 hr treatment for the RNA Polymerase I and III inhibitors, 5 hr treatment for the Cdk7 inhibitor). This experiment was conducted as in Figure 3A,B, but transcripts were detected with probe set designed against super-chromosomal family one sequences (SF1). (B) Quantification of smFISH foci from (A) after treatment of HeLa cells with small-molecule inhibitors against Cdk7, RNA Pol I and RNA Pol III. When compared to controls, the number of smFISH foci was substantially reduced after inhibition of RNA Pol II activator, Cdk7. Error bars represent the mean and standard deviation of at least 240 cells. Scale bar, 25 µm.