A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300

  1. May Khanna1,4,
  2. Harm Van Bakel1,4,
  3. Xinyi Tang1,
  4. John A. Calarco1,2,
  5. Tomas Babak1,2,
  6. Grace Guo1,
  7. Andrew Emili1,2,
  8. Jack F. Greenblatt1,2,
  9. Timothy R. Hughes1,2,
  10. Nevan J. Krogan3 and
  11. Benjamin J. Blencowe1,2
  1. 1Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada
  2. 2Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
  3. 3Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research, University of California at San Francisco, San Francisco, California 94158, USA
    1. 4 These authors contributed equally to this work.

    Abstract

    Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3′-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.

    Keywords:

    Keywords

    Footnotes

    • Reprint requests to: Benjamin J. Blencowe, Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, University of Toronto, Room 1016, 160 College Street, Ontario M5S 3E1, Canada; e-mail: b.blencowe{at}utoronto.ca; fax: (416) 946-5545.

    • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1790509.

      • Received June 20, 2009.
      • Accepted August 18, 2009.

    Related Article

    | Table of Contents