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Involvement of AGO1 and AGO2 in mammalian transcriptional silencing

Nature Structural & Molecular Biology volume 13, pages 787–792 (2006)Cite this article

Abstract

Duplex RNAs complementary to messenger RNA inhibit translation in mammalian cells by RNA interference (RNAi). Studies have reported that RNAs complementary to promoter DNA also inhibit gene expression. Here we show that the human homologs of Argonaute-1 (AGO1) and Argonaute-2 (AGO2) link the silencing pathways that target mRNA with pathways mediating recognition of DNA. We find that synthetic antigene RNAs (agRNAs) complementary to transcription start sites or more upstream regions of gene promoters inhibit gene transcription. This silencing occurs in the nucleus, requires high promoter activity and does not necessarily require histone modification. AGO1 and AGO2 associate with promoter DNA in cells treated with agRNAs, and inhibiting expression of AGO1 or AGO2 reverses transcriptional and post-transcriptional silencing. Our data indicate key linkages and important mechanistic distinctions between transcriptional and post-transcriptional silencing pathways in mammalian cells.

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Figure 1: Inhibition by agRNAs complementary to sequences upstream from the transcription start sites (+1) of genes encoding HTT, AR and PR.
Figure 2: Effects of high or low levels of basal gene expression on silencing by agRNAs and siRNAs.
Figure 3: Histone modification and activity of agRNAs.
Figure 4: Involvement of AGO1 and AGO2 in transcriptional silencing.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIGMS 60642 and 73042 to D.R.C., CA 10151 to K.E.H. and SPORE CA70907 to J.D.M.), the High-Q Foundation, the Robert A. Welch Foundation (I-1244 to D.R.C.) and the Gillson Longenbough Foundation (to J.D.M.). We thank A. Khvorova and Y. Fedorov (Dharmacon) for providing the pooled RNAs needed to silence AGO2, V. Pashkov for help with the nuclear run-on assay and K. Morris for advice and encouragement.

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Authors and Affiliations

  1. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, 75390-9041, Texas, USA

    Bethany A Janowski, Kenneth E Huffman, Jacob C Schwartz, Rosalyn Ram, Robert Nordsell, David S Shames, John D Minna & David R Corey

  2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, 75390-9041, Texas, USA

    Bethany A Janowski, Kenneth E Huffman, Jacob C Schwartz, Rosalyn Ram, Robert Nordsell & David R Corey

  3. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, 75390-9041, Texas, USA

    David S Shames & John D Minna

  4. Department of the Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, 75390-9041, Texas, USA

    David S Shames & John D Minna

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  1. Bethany A Janowski
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  2. Kenneth E Huffman
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Contributions

B.A.J., K.E.H, J.C.S and D.S.S. designed and performed experiments. R.R. and R.N. performed experiments. B.A.J., J.D.M. and D.R.C. supervised the experiments.

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Correspondence to Bethany A Janowski or David R Corey.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Western analysis showing inhibition of PR in MCF-7 cells by mismatch-containing RNA duplex MM1, agRNAs PR26 and PR24, and siRNAs PRrna1 and PRrna2. (PDF 71 kb)

Supplementary Fig. 2

Inhibition of AGO1 expression measured after a second transfection with an anti-Ago1 siRNA or a noncomplementary oligomer. (PDF 63 kb)

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Janowski, B., Huffman, K., Schwartz, J. et al. Involvement of AGO1 and AGO2 in mammalian transcriptional silencing. Nat Struct Mol Biol 13, 787–792 (2006). https://doi.org/10.1038/nsmb1140

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