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RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7

Abstract

At the 3′ ends of protein-coding genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine residues (Tyr1) of its C-terminal domain (CTD). In addition, the associated cleavage-and-polyadenylation factor (CPF) cleaves the transcript and adds a poly(a) tail. Whether these events are coordinated and how they lead to transcription termination remains poorly understood. Here we show that CPF from Saccharomyces cerevisiae is a Pol II–CTD phosphatase and that the CPF subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, the activity of Glc7 is required for normal Tyr1 dephosphorylation at the polyadenylation site, for recruitment of termination factors Pcf11 and Rtt103 and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing by CPF and transcription termination are coupled via Glc7-dependent Pol II–Tyr1 dephosphorylation.

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Figure 1: CPF subunit Glc7 is a Pol II–CTD Tyr1 phosphatase in vitro.
Figure 2: Glc7 is required for dephosphorylation of Tyr1 but not Ser2 in vivo.
Figure 3: Ssu72 does not change Tyr1 phosphorylation levels in vitro and in vivo.
Figure 4: Tyr1 dephosphorylation by Glc7 is required for normal termination-factor recruitment and transcription termination in vivo.

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Acknowledgements

We thank A. Cheung (Cramer laboratory), N.A. Yewdall (Passmore laboratory) and the mass spectrometry facility at the MRC Laboratory of Molecular Biology (LMB) for help, D. Barford for discussions, E. Kremmer and D. Eick (Helmholtz Zentrum München) for antibodies and S. Munro (MRC-LMB) for yeast strains. A.D.E. was supported by a Woolf Fisher Trust Scholarship, and K.W. was supported by a fellowship from the Deutsche Forschungsgemeinschaft. Work in the lab of L.A.P. is supported by MRC grant MC_U105192715 (L.A.P.) and European Research Council (ERC) Starting Grant 261151 (L.A.P.). P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB646, SFB960, SFB1064, CIPSM, NIM, QBM), an ERC Advanced Grant, the Jung-Stiftung and the Vallee Foundation.

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A.S., A.D.E., S.E. and K.W. performed experiments. M.L. analyzed data. L.A.P. and P.C. designed and supervised research. A.S., A.D.E., L.A.P. and P.C. wrote the manuscript.

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Correspondence to Patrick Cramer or Lori A Passmore.

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Integrated supplementary information

Supplementary Figure 1 Growth analysis and fluorescence microscopy of anchor-away yeast strains.

(a) Serial dilutions of wild-type and Glc7 and Ssu72 anchor-away yeast strains plated on YPD (left panel) and YPD + rapamycin (right panel) show that rapamycin is lethal for the anchor-away strains but it has no effect on wild-type growth. FRB, FKBP12-rapamycin-binding. (b) Fluorescence microscopy of fixed cells of the Ssu72-FRB/Glc7-mCherry strain shows that Glc7 is located in both cytoplasm and nucleus (left panel). This distribution does not change when rapamycin is added to the cells (lower panel). DAPI stain is shown as a control (right panel).

Supplementary Figure 2 Depletion of Glc7 from the nucleus leads to a defect in Tyr1-P dephosphorylation.

ChIP-chip occupancy profile of Tyr1-phosphorylated Pol II over 619 genes without and with rapamycin (solid and dotted line, respectively; see Fig. 2a for normalized profiles). The profiles include the region from 250 nucleotides upstream of the transcription start site (TSS) to 400 nucleotides downstream of the polyadenylation site (pA).

Supplementary Figure 3 Genome-wide ChIP occupancy of Tyr1-phosphorylated Pol II around the pA site is not influenced by rapamycin in wild-type yeast.

ChIP-chip occupancy profiling of Tyr1-phosphorylated Pol II over 619 genes aligned at the pA site (dashed line) and normalized against the corresponding Rpb3 profile without and with rapamycin (violet line and violet dotted line, respectively). The profile in a region from 400 nucleotides upstream to 400 nucleotides downstream of the polyA site is shown.

Supplementary Figure 4 Uncropped western blots of Pol II CTD in vitro assays.

(a) Uncropped blots of Figure 1b in the main text. (b) Uncropped blots of Figure 3a in the main text. See main text for details.

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Supplementary Figures 1–4 (PDF 2473 kb)

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Schreieck, A., Easter, A., Etzold, S. et al. RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7. Nat Struct Mol Biol 21, 175–179 (2014). https://doi.org/10.1038/nsmb.2753

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