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Fast ribozyme cleavage releases transcripts from RNA polymerase II and aborts co-transcriptional pre-mRNA processing

Nature Structural & Molecular Biology volume 16, pages 916–922 (2009)Cite this article

Abstract

We investigated whether a continuous transcript is necessary for co-transcriptional pre-mRNA processing. Cutting an intron with the fast-cleaving hepatitis δ ribozyme, but not the slower hammerhead, inhibited splicing. Therefore, exon tethering to RNA polymerase II (Pol II) cannot rescue splicing of a transcript severed by a ribozyme that cleaves rapidly relative to the rate of splicing. Ribozyme cutting also released cap-binding complex (CBC) from the gene, suggesting that exon 1 is not tethered. Unexpectedly, cutting within exons inhibited splicing of distal introns, where exon definition is not affected, probably owing to disruption of the interactions with the CBC and the Pol II C-terminal domain that facilitate splicing. Ribozyme cutting within the mRNA also inhibited 3′ processing and transcription termination. We propose that damaging the nascent transcript aborts pre-mRNA processing and that this mechanism may help to prevent association of processing factors with Pol II that is not productively engaged in transcription.

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Figure 1: Ribozyme cleavage in globin exon 1 or 2 inhibits splicing of introns 1 and 2 and 3′ end processing.
Figure 2: Ribozyme cleavage in exon 2 releases CBC from the gene.
Figure 3: Hepatitis δ but not hammerhead ribozyme cleavage in intron 2 inhibits splicing.
Figure 4: Hepatitis δ ribozyme cleavage in introns inhibits mRNA processing, protein expression and RNA editing.
Figure 5: Ribozyme cleavage in exon 3 inhibits splicing independently of effects on exon definition.
Figure 6: Inhibition of poly(A) site cleavage and transcription termination by ribozyme cleavage in exon 3.

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Acknowledgements

Supported by the US National Institutes of Health (NIH) grant GM58613 to D.B. We thank T. Blumenthal, R. Davis, R. Ferguson, J. Underwood, J. Manley, K. Ryman, B. Cullen and S. Chavez for helpful suggestions, E. Izaurralde (Universität Tubingen) for anti-CBP80, B. Cullen (Duke University) for miR21 and miR30 plasmids, J. Lykke-Andersen (University of Colorado) for Upf1 and PM-Scl75 plasmids, and the University of Colorado Health Sciences Center Cancer Center sequencing facility.

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  1. Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, University of Colorado at Denver Health Sciences Center, Aurora, Colorado, USA

    Nova Fong & David L Bentley

  2. Department of Molecular Biology and Functional Genomics, Stockholm University, Stockholm, Sweden

    Marie Öhman

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N.F., M.O. and D.L.B. designed experiments; N.F. performed the experiments, except the RNA editing analysis in Figure 4c, which was done by M.O.; D.L.B. wrote the manuscript.

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Correspondence to David L Bentley.

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Fong, N., Öhman, M. & Bentley, D. Fast ribozyme cleavage releases transcripts from RNA polymerase II and aborts co-transcriptional pre-mRNA processing. Nat Struct Mol Biol 16, 916–922 (2009). https://doi.org/10.1038/nsmb.1652

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