Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Exploring functional relationships between components of the gene expression machinery

Nature Structural & Molecular Biology volume 12, pages 175–182 (2005)Cite this article

Abstract

Eukaryotic gene expression requires the coordinated activity of many macromolecular machines including transcription factors and RNA polymerase, the spliceosome, mRNA export factors, the nuclear pore, the ribosome and decay machineries. Yeast carrying mutations in genes encoding components of these machineries were examined using microarrays to measure changes in both pre-mRNA and mRNA levels. We used these measurements as a quantitative phenotype to ask how steps in the gene expression pathway are functionally connected. A multiclass support vector machine was trained to recognize the gene expression phenotypes caused by these mutations. In several cases, unexpected phenotype assignments by the computer revealed functional roles for specific factors at multiple steps in the gene expression pathway. The ability to resolve gene expression pathway phenotypes provides insight into how the major machineries of gene expression communicate with each other.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Hierarchical clustering of intron accumulation indexes.
Figure 2: Multiclass SVM analysis of gene expression data.
Figure 3: Suppression of mex67-6 mRNA export defect by an spt4-null allele.
Figure 4: Ded1p associates with snRNAs in vitro.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Reed, R. Coupling transcription, splicing and mRNA export. Curr. Opin. Cell. Biol. 15, 326–331 (2003).

    CAS  PubMed  Google Scholar 

  2. Dimaano, C. & Ullman, K.S. Nucleocytoplasmic transport: integrating mRNA production and turnover with export through the nuclear pore. Mol. Cell. Biol. 24, 3069–3076 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Clark, T.A., Sugnet, C.W. & Ares, M. Jr. Genomewide analysis of mRNA processing in yeast using splicing-specific microarrays. Science 296, 907–910 (2002).

    CAS  PubMed  Google Scholar 

  4. Wang, Y. et al. Precision and functional specificity in mRNA decay. Proc. Natl. Acad. Sci. USA 99, 5860–5865 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Holstege, F.C. et al. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 (1998).

    CAS  PubMed  Google Scholar 

  6. Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M.D. & Hughes, T.R. Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol. Cell. Biol. 24, 5534–5547 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Hirose, Y., Tacke, R. & Manley, J.L. Phosphorylated RNA polymerase II stimulates pre-mRNA splicing. Genes Dev. 13, 1234–1239 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Zeng, C. & Berget, S.M. Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing. Mol. Cell. Biol. 20, 8290–8301 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Schwer, B. & Shuman, S. Conditional inactivation of mRNA capping enzyme affects yeast pre-mRNA splicing in vivo. RNA 2, 574–583 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Fresco, L.D. & Buratowski, S. Conditional mutants of the yeast mRNA capping enzyme show that the cap enhances, but is not required for, mRNA splicing. RNA 2, 584–596 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Chuang, R.Y., Weaver, P.L., Liu, Z. & Chang, T.H. Requirement of the DEAD-box protein ded1p for messenger RNA translation. Science 275, 1468–1471 (1997).

    CAS  PubMed  Google Scholar 

  12. Stutz, F. & Izaurralde, E. The interplay of nuclear mRNP assembly, mRNA surveillance and export. Trends. Cell Biol. 13, 319–327 (2003).

    CAS  PubMed  Google Scholar 

  13. Strasser, K. & Hurt, E. Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p. Nature 413, 648–652 (2001).

    CAS  PubMed  Google Scholar 

  14. Segref, A. et al. Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores. EMBO J. 16, 3256–3271 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Strasser, K. et al. TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417, 304–308 (2002).

    PubMed  Google Scholar 

  16. Herold, A., Teixeira, L. & Izaurralde, E. Genome-wide analysis of nuclear mRNA export pathways in Drosophila. EMBO J. 22, 2472–2483 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Rehwinkel, J. et al. Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster. Nat. Struct. Mol. Biol. 11, 558–566 (2004).

    CAS  PubMed  Google Scholar 

  18. Hartzog, G.A., Speer, J.L. & Lindstrom, D.L. Transcript elongation on a nucleoprotein template. Biochim. Biophys. Acta 1577, 276–286 (2002).

    CAS  PubMed  Google Scholar 

  19. Mueller, C.L. & Jaehning, J.A. Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex. Mol. Cell. Biol. 22, 1971–1980 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Squazzo, S.L. et al. The Paf1 complex physically and functionally associates with transcription elongation factors in vivo. EMBO J. 21, 1764–1774 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Krogan, N.J. et al. RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach. Mol. Cell. Biol. 22, 6979–6992 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Lindstrom, D.L. et al. Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol. Cell. Biol. 23, 1368–1378 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Kim, M., Ahn, S.H., Krogan, N.J., Greenblatt, J.F. & Buratowski, S. Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes. EMBO J. 23, 354–364 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Chang, M. et al. A complex containing RNA polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p plays a role in protein kinase C signaling. Mol. Cell. Biol. 19, 1056–1067 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kobor, M.S. & Greenblatt, J. Regulation of transcription elongation by phosphorylation. Biochim. Biophys. Acta 1577, 261–275 (2002).

    CAS  PubMed  Google Scholar 

  26. Lee, T.I. & Young, R.A. Transcription of eukaryotic protein-coding genes. Annu. Rev. Genet. 34, 77–137 (2000).

    CAS  PubMed  Google Scholar 

  27. Fischbeck, J.A., Kraemer, S.M. & Stargell, L.A. SPN1, a conserved gene identified by suppression of a postrecruitment-defective yeast TATA-binding protein mutant. Genetics 162, 1605–1616 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Skaar, D.A. & Greenleaf, A.L. The RNA polymerase II CTD kinase CTDK-I affects pre-mRNA 3′ cleavage/polyadenylation through the processing component Pti1p. Mol. Cell 10, 1429–1439 (2002).

    CAS  PubMed  Google Scholar 

  29. Ahn, S.H., Kim, M. & Buratowski, S. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3′ end processing. Mol. Cell 13, 67–76 (2004).

    CAS  PubMed  Google Scholar 

  30. Betz, J.L. et al. Phenotypic analysis of Paf1/RNA polymerase II complex mutations reveals connections to cell cycle regulation, protein synthesis, and lipid and nucleic acid metabolism. Mol. Genet. Genomics 268, 272–285 (2002).

    CAS  PubMed  Google Scholar 

  31. Brown, M.P. et al. Knowledge-based analysis of microarray gene expression data by using support vector machines. Proc. Natl. Acad. Sci. USA 97, 262–967 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Lee, Y. & Lee, C.K. Classification of multiple cancer types by multicategory support vector machines using gene expression data. Bioinformatics 19, 1132–1139 (2003).

    CAS  PubMed  Google Scholar 

  33. Furey, T.S. et al. Support vector machine classification and validation of cancer tissue samples using microarray expression data. Bioinformatics 16, 906–914 (2000).

    CAS  PubMed  Google Scholar 

  34. Mnaimneh, S. et al. Exploration of essential gene functions via titratable promoter alleles. Cell 118, 31–44 (2004).

    CAS  PubMed  Google Scholar 

  35. Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25–29 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Ramaswamy, S. et al. Multiclass cancer diagnosis using tumor gene expression signatures. Proc. Natl. Acad. Sci. USA 98, 15149–15154 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Pannone, B.K., Kim, S.D., Noe, D.A. & Wolin, S.L. Multiple functional interactions between components of the Lsm2–Lsm8 complex, U6 snRNA, and the yeast La protein. Genetics 158, 187–196 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Bouveret, E., Rigaut, G., Shevchenko, A., Wilm, M. & Seraphin, B. A Sm-like protein complex that participates in mRNA degradation. EMBO J. 19, 1661–1671 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Tharun, S. et al. Yeast Sm-like proteins function in mRNA decapping and decay. Nature 404, 515–518 (2000).

    CAS  PubMed  Google Scholar 

  40. Izaurralde, E. et al. A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell 78, 657–668 (1994).

    CAS  PubMed  Google Scholar 

  41. Lewis, J.D., Gorlich, D. & Mattaj, I.W. A yeast cap binding protein complex (yCBC) acts at an early step in pre-mRNA splicing. Nucleic Acids Res. 24, 3332–3336 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Andrulis, E.D. et al. The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila. Nature 420, 837–841 (2002).

    CAS  PubMed  Google Scholar 

  43. Libri, D., Graziani, N., Saguez, C. & Boulay, J. Multiple roles for the yeast SUB2/yUAP56 gene in splicing. Genes Dev. 15, 36–41 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Kistler, A.L. & Guthrie, C. Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase. Genes Dev. 15, 42–49 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Fleckner, J., Zhang, M., Valcarcel, J. & Green, M.R. U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev. 11, 1864–1872 (1997).

    CAS  PubMed  Google Scholar 

  46. Jensen, T.H., Boulay, J., Rosbash, M. & Libri, D. The DECD box putative ATPase Sub2p is an early mRNA export factor. Curr. Biol. 11, 1711–1715 (2001).

    CAS  PubMed  Google Scholar 

  47. Gatfield, D. et al. The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr. Biol. 11, 1716–1721 (2001).

    CAS  PubMed  Google Scholar 

  48. Zenklusen, D., Vinciguerra, P., Wyss, J.C. & Stutz, F. Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p. Mol. Cell. Biol. 22, 8241–8253 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Rondon, A.G., Jimeno, S., Garcia-Rubio, M. & Aguilera, A. Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation. J. Biol. Chem. 278, 39037–39043 (2003).

    CAS  PubMed  Google Scholar 

  50. Jamieson, D.J., Rahe, B., Pringle, J. & Beggs, J.D. A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase. Nature 349, 715–717 (1991).

    CAS  PubMed  Google Scholar 

  51. Stevens, S.W. et al. Composition and functional characterization of the yeast spliceosomal penta-snRNP. Mol. Cell 9, 31–44 (2002).

    CAS  PubMed  Google Scholar 

  52. Zhou, Z., Licklider, L.J., Gygi, S.P. & Reed, R. Comprehensive proteomic analysis of the human spliceosome. Nature 419, 182–185 (2002).

    CAS  PubMed  Google Scholar 

  53. Ares, M., Jr., Grate, L. & Pauling, M.H. A handful of intron-containing genes produces the lion's share of yeast mRNA. RNA 5, 1138–1139 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Shevchenko, A., Schaft, D., Roguev, A., Pijnappel, W.W. & Stewart, A.F. Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective. Mol. Cell. Proteomics 1, 204–212 (2002).

    CAS  PubMed  Google Scholar 

  55. Legrain, P. & Selig, L. Genome-wide protein interaction maps using two-hybrid systems. FEBS Lett. 480, 32–36 (2000).

    CAS  PubMed  Google Scholar 

  56. Zhong, J., Zhang, H., Stanyon, C.A., Tromp, G. & Finley, R.L. Jr. A strategy for constructing large protein interaction maps using the yeast two-hybrid system: regulated expression arrays and two-phase mating. Genome Res. 13, 2691–2699 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Tong, A.H. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364–2368 (2001).

    CAS  PubMed  Google Scholar 

  58. Dudoit, S., Gentleman, R.C. & Quackenbush, J. Open source software for the analysis of microarray data. Biotechniques (suppl.), 45–51 (2003).

  59. Yang, Y.H. et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15 (2002).

    PubMed  PubMed Central  Google Scholar 

  60. Sturn, A., Quackenbush, J. & Trajanoski, Z. Genesis: cluster analysis of microarray data. Bioinformatics 18, 207–208 (2002).

    CAS  PubMed  Google Scholar 

  61. Pavlidis, P., Wapinski, I. & Noble, W.S. Support vector machine classification on the web. Bioinformatics 20, 586–587 (2004).

    CAS  PubMed  Google Scholar 

  62. Cheng, S.C., Newman, A.N., Lin, R.J., McFarland, G.D. & Abelson, J.N. Preparation and fractionation of yeast splicing extract. Methods Enzymol. 181, 89–96 (1990).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank B. Noble for help with SVM analysis, S. Ruby, P. Silver, E. Hurt and L. Hicke for yeast strains, and A. Caroll and J. DeRisi for advice and assistance with microarrays. This work was primarily funded by grants to M.A. from the US National Institutes of Health (NIH) (GM040478), the Packard Foundation, and the W.M. Keck Foundation (to the University of California Santa Cruz RNA Center). The Howard Hughes Medical Institute Professors program supported Y.M.-G. Grants to G.H. from the NIH (GM060479), and the University of California Cancer Research Coordinating Committee supported this work as well, and T.-H.C. was supported by the NIH (GM48752) and the US National Science Foundation (MCB-9982726).

Author information

Author notes

  1. Yael Mandel-Gutfreund

    Present address: Faculty of Biology, Technion, Haifa, 32000, Israel

  2. Tyson A Clark

    Present address: Affymetrix, 3380 Central Expressway, Santa Clara, California, 95051, USA

  3. Todd Burckin and Roland Nagel: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular, Cell & Developmental Biology, University of California Santa Cruz, Santa Cruz, 95064, California, USA

    Todd Burckin, Roland Nagel, Yael Mandel-Gutfreund, Tyson A Clark, Sharon Squazzo, Grant Hartzog & Manuel Ares Jr

  2. Center for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, 95064, California, USA

    Roland Nagel, Yael Mandel-Gutfreund & Manuel Ares Jr

  3. Center for Biomolecular Science and Engineering, University of California Santa Cruz, Santa Cruz, 95064, California, USA

    Lily Shiue, Grant Hartzog & Manuel Ares Jr

  4. Department of Molecular Genetics, The Ohio State University, 484 West 12th Avenue, Columbus, 43210, Ohio, USA

    Jean-Leon Chong & Tien-Hsien Chang

Authors
  1. Todd Burckin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roland Nagel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yael Mandel-Gutfreund
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lily Shiue
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tyson A Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jean-Leon Chong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tien-Hsien Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sharon Squazzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Grant Hartzog
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Manuel Ares Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Manuel Ares Jr.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Comparison of microarray with QPCR. (PDF 83 kb)

Supplementary Fig. 2

Intron versus exon log ratios. (PDF 111 kb)

Supplementary Fig. 3

Ceg1 phenotype. (PDF 86 kb)

Supplementary Fig. 4

ROC plots for the SVM results. (PDF 63 kb)

Supplementary Table 1

Values for graph in Supplementary Figure 1. (PDF 47 kb)

Supplementary Table 2

Oligonucleotides used for QPCR analysis. (PDF 44 kb)

Supplementary Table 3

Intron accumulation index data for clustering. (TXT 193 kb)

Supplementary Table 4

Multiclass SVM scores. (XLS 33 kb)

Supplementary Table 5

Strain and experimental details. (XLS 32 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Burckin, T., Nagel, R., Mandel-Gutfreund, Y. et al. Exploring functional relationships between components of the gene expression machinery. Nat Struct Mol Biol 12, 175–182 (2005). https://doi.org/10.1038/nsmb891

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb891

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing