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Bimodal expression of PHO84 is modulated by early termination of antisense transcription

Abstract

Many Saccharomyces cerevisiae genes encode antisense transcripts, some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense (AS) RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single-molecule resolution fluorescent in situ hybridization (smFISH) to investigate antisense-mediated transcription regulation. We show that PHO84 AS RNA acts as a bimodal switch, in which continuous, low-frequency antisense transcription represses sense expression within individual cells. Surprisingly, antisense RNAs do not accumulate at the PHO84 gene but are exported to the cytoplasm. Furthermore, rather than stabilizing PHO84 AS RNA, the loss of Rrp6 favors its elongation by reducing early transcription termination by Nrd1–Nab3–Sen1. These observations suggest that PHO84 silencing results from antisense transcription through the promoter rather than the static accumulation of antisense RNAs at the repressed gene.

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Figure 1: PHO84 sense and antisense expression are anticorrelated.
Figure 2: PHO84 AS RNAs do not accumulate at the PHO84 locus.
Figure 3: PHO84 antisense RNAs are polyadenylated by Pap1.
Figure 4: PHO84 antisense nuclear detection needs ongoing transcription.
Figure 5: Effect of Δrrp6 on antisense RNA half-life and transcription.
Figure 6: PHO84 antisense transcription is attenuated by Nrd1–Nab3–Sen1 (NNS).

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Acknowledgements

We thank T.H. Jensen (Aarhus University, Denmark), D. Libri (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France), A. Morillon (Curie Institute, Paris) and D. Tollervey (University of Edinburgh, UK) for strains; K. Weis (University of California, Berkeley) for communicating data before publication; D. Larson (National Cancer Institute, Bethesda, Maryland) for updates of image analysis software; and M. Oeffinger, F. Robert, O. Gahura, A. Maffioletti, C. Dargemont, D. Libri and V. Géli for discussions and critical reading of the manuscript. This work was supported by SystemsX and Novartis fellowships (M.C.); an EMBO fellowship (E.G.); the Swiss National Science Foundation (grant no. 31003A_130292) and National Center of Competence in Research “Frontiers in Genetics,” IGE3 and the Canton of Geneva (F.S.); and the Canadian Institutes of Health Research (MOP-BMB-232642), the Canadian Foundation for Innovation and the 'Fonds de recherche du Québec–Santé' (Chercheur Boursier Junior I) (D.Z.).

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M.C. performed ChIP and RNA analyses; S.R. performed the smFISH experiments; E.G. prepared strains and performed RNA analyses; V.I. made mutants and RNA analyses. M.C., S.R., D.Z. and F.S. analyzed that data; D.Z. and F.S. supervised the project and wrote the manuscript.

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Correspondence to Françoise Stutz or Daniel Zenklusen.

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The authors declare no competing financial interests.

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Castelnuovo, M., Rahman, S., Guffanti, E. et al. Bimodal expression of PHO84 is modulated by early termination of antisense transcription. Nat Struct Mol Biol 20, 851–858 (2013). https://doi.org/10.1038/nsmb.2598

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