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The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II

Nature volume 432, pages 517–522 (2004)Cite this article

A Corrigendum to this article was published on 10 February 2005

Abstract

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNApII) largest subunit consists of multiple heptapeptide repeats with the consensus sequence YSPTSPS. Different CTD phosphorylation patterns act as recognition sites for the binding of various messenger RNA processing factors, thereby coupling transcription and mRNA processing1. Polyadenylation factors are co-transcriptionally recruited by phosphorylation of CTD serine 2 (ref. 2) and these factors are also required for transcription termination3,4. RNApII transcribes past the poly(A) site, the RNA is cleaved by the polyadenylation machinery, and the RNA downstream of the cleavage site is degraded. Here we show that Rtt103 and the Rat1/Rai1 5′ → 3′ exonuclease are localized at 3′ ends of protein coding genes. In rat1-1 or rai1Δ cells, RNA 3′ to polyadenylation sites is greatly stabilized and termination defects are seen at many genes. These findings support a model in which poly(A) site cleavage and subsequent degradation of the 3′-downstream RNA by Rat1 trigger transcription termination5,6.

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Figure 1: Isolation of a complex containing Rtt103, RNA polymerase II and the Rat1/Rai1 exonuclease.
Figure 2: Rtt103 and Rat1/Rai1 localize at 3′ ends of genes.
Figure 3: Rat1 degrades 3′-downstream RNA but is not required for mRNA cleavage or polyadenylation.
Figure 4: Rat1/Rai1 promotes transcription termination.

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Acknowledgements

We thank C. Moore and her laboratory for advice on polyadenylation reactions; N. Proudfoot, S. McCracken and B. Blencowe for sharing unpublished information; and the Taplin Mass Spectroscopy facility at Harvard Medical School and D. Richards of Affinium Pharmaceuticals for mass spectroscopy. N.J.K. was supported by a Doctoral Fellowship from the Canadian Institutes of Health Research (CIHR). O.J.R. is supported by funding from the Bauer Center for Genomics Research. This research was supported by grants to S.B. from the US National Institutes of Health, and to J.F.G. from the Canadian Institutes of Health Research, the Ontario Genomics Institute, and the National Cancer Institute of Canada with funds from the Canadian Cancer Society. L.V. is a Fellow and S.B. is a Scholar of the Leukemia and Lymphoma Society.Authors’ contributions All authors contributed to the conception and design of the experiments. M.K. performed the CTD affinity purification of Rtt103, RT–PCR analysis, ChIP analyses and Rat1 mutagenesis. N.K. did the Rtt103-TAP purification and the SGA analysis. L.V. conducted the immunoblotting, exonuclease and polyadenylation assays. E.N. performed the Pcf11 and Rna15 TAP purifications. The microarray experiments were done by M.K. and O.J.R. The paper was written by M.K., J.G. and S.B. with input from the other authors.

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Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts, 02115, USA

    Minkyu Kim, Lidia Vasiljeva & Stephen Buratowski

  2. Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario, M5G 1L6, Canada

    Nevan J. Krogan, Eduard Nedea & Jack F. Greenblatt

  3. Bauer Center for Genomics Research, Harvard University, 7 Divinity Ave., Cambridge, Massachusetts, 02138, USA

    Oliver J. Rando

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  1. Minkyu Kim
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Correspondence to Stephen Buratowski.

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Supplementary information

Supplementary Figures 1 and 2

Fig 1: The termination defect in the rat1-1 mutant is not due to protein instability or defects in polyadenylation; Fig 2: The Rat1 D235A mutant lacks exonuclease activity. (DOC 81 kb)

Supplementary Table 1

The Rat1 D235A mutant lacks exonuclease activity. (DOC 26 kb)

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Kim, M., Krogan, N., Vasiljeva, L. et al. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432, 517–522 (2004). https://doi.org/10.1038/nature03041

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