Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis
- 1Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom;
- 2Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia 22908, USA
Abstract
Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5–Air1/Air2–Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 5′ region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 5′ region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 5′ region of the rDNA, imposing persistent transcription blocks when RNase H is limiting.
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Footnotes
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↵3 Corresponding author.
E-MAIL d.tollervey{at}ed.ac.uk; FAX 44-131-650-7040.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.573310.
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Supplemental material is available at http://www.genesdev.org.
- Received December 22, 2009.
- Accepted May 18, 2010.
- Copyright © 2010 by Cold Spring Harbor Laboratory Press
Freely available online through the Genes & Development Open Access option.