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Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells

Abstract

We show that convergent transcription induces transcriptional gene silencing (TGS) in trans for both fission yeast and mammalian cells. This method has advantages over existing strategies to induce gene silencing. Previous studies in fission yeast have characterized TGS as a cis-specific process involving RNA interference that maintains heterochromatic regions such as centromeres. In contrast, in mammalian cells, gene silencing is known to occur through a post-transcriptional mechanism that uses exogenous short interfering RNAs or endogenous microRNAs to inactivate mRNA. We now show that the introduction of convergent transcription plasmids into either Schizosaccharomyces pombe or mammalian cells allows the production of double-stranded RNA from inserted gene fragments, resulting in TGS of endogenous genes. We predict that using convergent transcription to induce gene silencing will be a generally useful strategy and allow for a fuller molecular understanding of the biology of TGS.

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Figure 1: S. pombe transformed convergent transcription plasmids are silenced by RNAi.
Figure 2: CTura4 plasmids induce TGS of endogenous ura4 in S. pombe.
Figure 3: Integrated convergent transcription constructs promote endogenous ura4 TGS in trans.
Figure 4: Transfection of mammalian CTura4 plasmid induces ACTG1 TGS.
Figure 5: In vitro and in vivo analysis of nuclear dicer activity.
Figure 6: Specificity of TGS induction by CTγACT1 transfection.
Figure 7: Spatial and temporal properties of plasmid-induced TGS.

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Acknowledgements

We thank T. Gligoris for advice and encouragement, E. White for help with HeLa cell culture and J. Monks for cloning. This work was supported by grants from Cancer Research UK and the Wellcome Trust to N.J.P. and by L'Oreal/UNESCO woman in science UK and Ireland award to M.G.

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M.G. performed all the experimental analyses. M.G. and N.J.P. designed the experiments and wrote the manuscript.

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Correspondence to Monika Gullerova or Nick J Proudfoot.

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Convergent transcription–mediated TGS as described in these studies is the subject of worldwide patent WO/2012/114111 held by ISIS Innovation of Oxford University.

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Gullerova, M., Proudfoot, N. Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells. Nat Struct Mol Biol 19, 1193–1201 (2012). https://doi.org/10.1038/nsmb.2392

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