Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis.
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.
El Hage
Aziz
A
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
French
Sarah L
SL
Beyer
Ann L
AL
Tollervey
David
D
D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D016914:Ribonuclease H / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
D012322:RNA Precursors; D012337:RNA, Ribosomal, 18S; D016914:Ribonuclease H; D004264:DNA Topoisomerases, Type I