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The proteomes of transcription factories containing RNA polymerases I, II or III

Nature Methods volume 8, pages 963–968 (2011)Cite this article

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Abstract

Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.

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Figure 1: Purification procedure.
Figure 2: Resolving different polymerases in native two-dimensional gels (run-ons in [32P]UTP are included).
Figure 3: The content of complexes I, II and III as determined by mass spectrometry.
Figure 4: Isolated complexes remain associated with DNA sequences found in vivo.

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References

  1. Roeder, R.G. The eukaryotic transcriptional machinery: complexities and mechanisms unforeseen. Nat. Med. 9, 1239–1244 (2003).

    Article  CAS  Google Scholar 

  2. Cramer, P. et al. Structure of eukaryotic RNA polymerases. Annu. Rev. Biophys. 37, 337–352 (2008).

    Article  CAS  Google Scholar 

  3. Das, R. et al. SR proteins function in coupling RNAP II transcription to pre-mRNA splicing. Mol. Cell 26, 867–881 (2007).

    Article  CAS  Google Scholar 

  4. Shi, Y. et al. Molecular architecture of the human pre-mRNA 3′ processing complex. Mol. Cell 33, 365–376 (2009).

    Article  CAS  Google Scholar 

  5. Cook, P.R. A model for all genomes; the role of transcription factories. J. Mol. Biol. 395, 1–10 (2010).

    Article  CAS  Google Scholar 

  6. Chakalova, L. & Fraser, P. Organization of transcription. Cold Spring Harb. Perspect. Biol. 2, a000729 (2010).

    Article  Google Scholar 

  7. Sutherland, H. & Bickmore, W.A. Transcription factories: gene expression in unions? Nat. Rev. Genet. 10, 457–466 (2009).

    Article  CAS  Google Scholar 

  8. Jackson, D.A., Iborra, F.J., Manders, E.M.M. & Cook, P.R. Numbers and organization of RNA polymerases, nascent transcripts and transcription units in HeLa nuclei. Mol. Biol. Cell 9, 1523–1536 (1998).

    Article  CAS  Google Scholar 

  9. Kimura, H., Tao, Y., Roeder, R.G. & Cook, P.R. Quantitation of RNA polymerase II and its transcription factors in an HeLa cell: little soluble holoenzyme but significant amounts of polymerases attached to the nuclear substructure. Mol. Cell. Biol. 19, 5383–5392 (1999).

    Article  CAS  Google Scholar 

  10. Jackson, D.A. & Cook, P.R. Transcription occurs at a nucleoskeleton. EMBO J. 4, 919–925 (1985).

    Article  CAS  Google Scholar 

  11. Ahmad, Y., Boisvert, F.M., Gregor, P., Cobley, A. & Lamond, A.I. NOPdb: Nucleolar Proteome Database–2008 update. Nucleic Acids Res. 37, D181–D184 (2009).

    Article  CAS  Google Scholar 

  12. Eskiw, C.H., Rapp, A., Carter, D.R.F. & Cook, P.R. RNA polymerase II activity is located on the surface of ∼87 nm protein-rich transcription factories. J. Cell Sci. 121, 1999–2007 (2008).

    Article  CAS  Google Scholar 

  13. Novakova, Z., Man, P., Novak, P., Hozak, P. & Hodny, Z. Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis. Electrophoresis 2, 1277–1287 (2006).

    Article  Google Scholar 

  14. Trudgian, D.C. et al. CPFP – The Oxford Central Proteomics Facility Pipeline. Clin. Proteomics 5 (suppl. 1), 94 (2009).

    Google Scholar 

  15. Griffin, N.M. et al. Label-free, normalized quantification of complex mass spectrometry data for proteomic analysis. Nat. Biotechnol. 28, 83–89 (2010).

    Article  CAS  Google Scholar 

  16. Hopper, A.K., Pai, D.A. & Engelke, D.R. Cellular dynamics of tRNAs and their genes. FEBS Lett. 584, 310–317 (2010).

    Article  CAS  Google Scholar 

  17. Iborra, F.J., Escargueil, A.E., Kwek, K.Y., Akoulitchev, A. & Cook, P.R. Molecular cross-talk between the transcription, translation, and nonsense-mediated decay machineries. J. Cell Sci. 117, 899–906 (2004).

    Article  CAS  Google Scholar 

  18. Dekker, J., Rippe, K., Dekker, M. & Kleckner, N. Capturing chromosome conformation. Science 295, 1306–1311 (2002).

    Article  CAS  Google Scholar 

  19. Cullen, K.E., Kladde, M.P. & Seyfred, M.A. Interaction between transcription regulatory regions of prolactin chromatin. Science 261, 203–206 (1993).

    Article  CAS  Google Scholar 

  20. Papantonis, A. et al. Active RNA polymerases: mobile or immobile molecular machines? PLoS Biol. 8, e1000419 (2010).

    Article  Google Scholar 

  21. Zheng, B., Han, M., Bernier, M. & Wen, J.K. Nuclear actin and actin-binding proteins in the regulation of transcription and gene expression. FEBS J. 276, 2669–2685 (2009).

    Article  CAS  Google Scholar 

  22. Hou, C. & Corces, V.G. Nups take leave of the nuclear envelope to regulate transcription. Cell 140, 306–308 (2010).

    Article  CAS  Google Scholar 

  23. Nadano, D., Aoki, C., Yoshinaka, T., Irie, S. & Sato, T.A. Electrophoretic characterization of ribosomal subunits and protein in apoptosis: specific downregulation of S11 in staurosporine-treated human breast carcinoma cells. Biochemistry 40, 15184–15193 (2001).

    Article  CAS  Google Scholar 

  24. Maclean, B., Eng, J.K., Beavis, R.C. & McIntosh, M. General framework for developing and evaluating database scoring algorithms using the TANDEM search engine. Bioinformatics 22, 2830–2832 (2006).

    Article  CAS  Google Scholar 

  25. Geer, L.Y. et al. Open mass spectrometry search algorithm. J. Proteome Res. 3, 958–964 (2004).

    Article  CAS  Google Scholar 

  26. Keller, A., Nesvizhskii, A.I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002).

    Article  CAS  Google Scholar 

  27. Shteynberg, D. et al. iProphet: Improved validation of peptide and protein IDs in the trans-proteomic pipeline. Poster session at: HUPO 7th Annual World Congress (August 16–20, Amsterdam 2008).

  28. Elias, J.E. & Gygi, S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).

    Article  CAS  Google Scholar 

  29. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57, 289–300 (1995).

    Google Scholar 

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Acknowledgements

We thank J. Bartlett for technical assistance, M. Vigneron (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg) for the 7C2 antibody, B. Thomas, D. Trudgian, G. Ridlova and M. Dreger for help with proteomics, M. Shaw for help with electron microscopy, and the Medical Research Council (S.M. and B.D.), EP Abraham Research Fund (B.D.), Biotechnology and Biological Sciences Research Council (A.P.), Wellcome Trust (A.P.) and Felix Scholarship Trust of Oxford University (S.B.) for support.

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Author notes

  1. Svitlana Melnik

    Present address: Present address: Division of Molecular Biology of the Cell II, German Cancer Research Center, Heidelberg, Germany.,

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Svitlana Melnik, Binwei Deng, Argyris Papantonis, Sabyasachi Baboo & Peter R Cook

  2. Leeds Institute of Molecular Medicine, University of Leeds, St. James's Hospital, Leeds, UK

    Ian M Carr

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Contributions

Experiments were designed by S.M., B.D., A.P., S.B. and P.R.C. S.M. developed the isolation procedure and carried out many of the validation experiments, S.M. and B.D. performed gel electrophoreses and mass spectrometry, A.P. developed native 3C and carried out RT-PCR, S.B. did the light microscopy, and I.M.C. developed software. All authors wrote the paper.

Corresponding author

Correspondence to Peter R Cook.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–3, Supplementary Note (PDF 1947 kb)

Supplementary Table 4

Experiment 1: output from the CPFP (FDRs for complexes I, II and III were 0.84, 0.8 and 0.82% respectively), and results from the SI analysis. (XLSX 1665 kb)

Supplementary Table 5

Experiment 2: output from the CPFP (FDRs for complexes I, II and III were all 0.8%). (XLSX 103 kb)

Supplementary Table 6

Experiment 3: output from the CPFP (FDRs for complexes II and III were 0.75 and 0.65%, respectively), and results from the SI analysis. (XLSX 243 kb)

Supplementary Table 7

Comparison of the proteomes seen in all three experiments. (XLSX 1036 kb)

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Melnik, S., Deng, B., Papantonis, A. et al. The proteomes of transcription factories containing RNA polymerases I, II or III. Nat Methods 8, 963–968 (2011). https://doi.org/10.1038/nmeth.1705

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