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B2 RNA binds directly to RNA polymerase II to repress transcript synthesis

Abstract

B2 RNA is a small noncoding RNA polymerase III transcript that represses mRNA transcription in response to heat shock in mouse cells. Here we define the mechanism by which B2 RNA inhibits RNA polymerase II (Pol II) transcription. Using a purified Pol II transcription system, we found that B2 RNA potently inhibits transcription by binding to core Pol II with high affinity and specificity. Through this interaction, B2 RNA assembles into preinitiation complexes at the promoter and blocks RNA synthesis. Once B2 RNA is removed from preinitiation complexes, transcriptional activity is restored. Our studies describe a previously unobserved mechanism of transcriptional repression by a small RNA and suggest that B2 RNA associates with Pol II at promoters in heat shocked cells to actively inhibit transcription.

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Figure 1: B2 RNA represses mRNA transcription with specificity in vitro.
Figure 2: B2 RNA targets Pol II to inhibit transcription.
Figure 3: B2 RNA binds Pol II with high affinity, specificity and kinetic stability.
Figure 4: B2 RNA binds a previously identified RNA docking site on Pol II.
Figure 5: B2 RNA prevents Pol II from forming functional initiation complexes.
Figure 6: B2 RNA associates with preinitiation complexes.
Figure 7: The effects of B2 RNA on migration of preinitiation complexes and transcriptional activity can be reversed with RNase treatment.

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Acknowledgements

We thank E. Podell for purified group I intron RNA. This research was supported by research project grant RPG-00-271-01-MGO from the American Cancer Society, public health service grant GM-68414 from the US National Institutes of Health and a Pew Scholarship in the Biomedical Sciences to J.A.G.

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Correspondence to Jennifer F Kugel or James A Goodrich.

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Espinoza, C., Allen, T., Hieb, A. et al. B2 RNA binds directly to RNA polymerase II to repress transcript synthesis. Nat Struct Mol Biol 11, 822–829 (2004). https://doi.org/10.1038/nsmb812

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