Figure 1. RNA associates with the pericentric regions of human mitotic chromosomes.

(A) RNA localization on human mitotic chromosomes. A schematic of the chromosome-associated RNA labeling approach is shown at top. Mitotic HeLa cells were spun onto coverslips and stained for DNA (blue), ethynyl uridine labeled RNA (EU-RNA, green), and H3K9me3 (red) or CENP-T (red) to mark centromeres. Top row of images: cells were not treated with EU (-EU). Bottom row of images: cells were labeled with EU for 12 hr (+EU). (B) α-satellite RNA localization on mitotic chromosomes. Mitotic DLD-1 cells were spun onto coverslips, then α-satellite RNA was detected with a probe recognizing the pericentric D1Z5 array on human chromosome 1 (green). Chromosomes were also stained for DNA (blue), H3K9me3 (red), and HEC1 (red) antibodies to mark pericentric heterochromatin and the core centromere/kinetochore region. (C) D1Z5 α-satellite RNA overlaps with pericentric heterochromatin, but not the core centromere/kinetochore. α-satellite RNA FISH with the D1Z5 probe (green) on a stretched DLD-1 mitotic chromosome, co-stained for DNA (blue), H3K9me3 (red), and HEC1 (red). Line scans show α-satellite RNA overlaps with H3K9me3, but not HEC1. (D) RNase sensitivity of D1Z5 α-satellite RNA FISH signal. Spread mitotic DLD-1 cells were treated ± RNases, then stained for DNA (blue), D1Z5 α-satellite RNA (green), and H3K9me3 (red). See also Figure 1—figure supplement 1 and 2.

DOI: http://dx.doi.org/10.7554/eLife.25299.003

Figure 1—figure supplement 1. Characterization of chromosome-associated pericentric RNA.

(A) RNase sensitivity of pericentric RNA. EU-labeled mitotic HeLa cells were spun onto coverslips and incubated with or without RNase A, RNase H, or RNase III as indicated, then stained for DNA (blue), EU-RNA (green) and CENP-A (red) to mark centromeres. (B, C) Representative images of different types of human cell lines, showing RNA localization on mitotic chromosomes. Cells were labeled with EU for 12 hr, then mitotic cells were spun onto coverslips and stained for DNA (blue), EU-RNA (green), and H3K9me3 (red). (B) shows cell lines tested that, like HeLa cells, show RNA concentrated around centromeres. (C) shows cell lines tested that show no apparent concentration of RNA around centromeres. (D) EU-RNA staining on DLD-1 chromosomes. Cells were labeled with EU and stained as described above. Although α-satellite RNA is detected on DLD-1 chromosomes by RNA FISH, there is little detectable EU-RNA signal. (E) β-satellite and Satellite III DNA and RNA on human mitotic chromosomes. Mitotic DLD-1 cells were spread onto coverslips, RNA or DNA FISH was performed to detect Satellite III (green) and β-satellite (red) sequences, and then chromosomes were stained for DNA (blue). The β-satellite probe recognizes sequences on chromosomes 13, 14, 15, 21, and 22, and the Satellite III probe recognizes sequences on chromosomes 14 and 22. (F) RNA FISH for D1Z5, a chromosome 1 specific α-satellite array, on HeLa mitotic chromosomes. Mitotic HeLa cells were spread onto coverslips, RNA FISH was performed to detect chromosome 1-specific D1Z5 α-satellite sequences (green), and chromosomes were stained for HEC1 to mark centromeres (red) and with Hoechst to stain for DNA (blue). (G) RNA FISH on DLD-1 chromosomes with a probe specific to an α-satellite array on chromosomes 13 and 21. Mitotic DLD-1 cells were spread onto coverslips, RNA FISH was performed to detect α-satellite 13/21 (green), and then chromosomes were stained for DNA (blue) and H3K9me3 (red). Line scans of all four 13 and 21 chromosomes show localization of α-satellite RNA and H3K9me3. White arrows delineate direction of line scan, and the labeling of homologous chromosomes as ‘homolog 1’ or ‘homolog 2’ is arbitrary. The Y-axis represents the pixel intensity along the drawn line.

DOI: http://dx.doi.org/10.7554/eLife.25299.004

Figure 1—figure supplement 2. Identifying the transcriptional requirements for chromosome-associated RNA.

(A) Representative images showing chromosome-associated EU-RNA signal after inhibiting RNA polymerases. HeLa cells were incubated with 0.5 mM EU and 50 μg/mL α-amanitin, 1 μM triptolide, 50 ng/mL actinomycin D, or 1 μM CX-5461 for 6 hr, then mitotic cells were spun onto coverslips and stained for DNA (blue), EU-RNA (green), and HEC1 to mark centromeres (red). (B) Quantificaton of EU-RNA at pericentric regions and RT-qPCR controls to assess inhibition of transcription by different RNA polymerase inhibitors. Far left panel: images from the experiment shown in A were quantified using pericentromere finder software and HEC1 staining as a centromere marker. Shown are the means of 3 separate experiments, 15 images quantified per condition per experiment. Four rightmost panels: total RNA was purified from cells treated with the indicated polymerase inhibitors (same cells shown in A), and RT-qPCR was performed with primers for control RNAs to check for inhibition of specific polymerases and to measure α-satellite RNA levels. All values were normalized to GAPDH RNA levels. Shown are the means of 3 separate experiments. All error bars represent standard error. (C) Diagram describing EU pulse/chase experiment to assess when chromosome-associated RNA is being transcribed. HeLa cells were incubated with 2 mM thymidine for 19 hr, then thymidine was washed out and 0.5 mM EU was added at the indicated time intervals before mitotic shake off. (D) Resulting mitotic spreads from experiment outlined in C, showing that chromosome-associated RNA is transcribed in the few hours before mitosis. Cells were harvested by mitotic shake off, spun onto coverslips, and stained for DNA (blue), and EU-RNA (green).

DOI: http://dx.doi.org/10.7554/eLife.25299.005