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Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry.

Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.

Pubmed ID: 11101529


  • Ajuh P
  • Kuster B
  • Panov K
  • Zomerdijk JC
  • Mann M
  • Lamond AI


The EMBO journal

Publication Data

December 1, 2000

Associated Grants


Mesh Terms

  • Adenosine Triphosphate
  • Amino Acid Sequence
  • Blotting, Western
  • Catalysis
  • Cell Cycle Proteins
  • Cell Nucleus
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA, Complementary
  • Databases, Factual
  • Electrophoresis, Polyacrylamide Gel
  • HeLa Cells
  • Humans
  • Models, Biological
  • Molecular Sequence Data
  • Precipitin Tests
  • RNA Splicing
  • RNA, Messenger
  • Recombinant Proteins
  • Ribosomes
  • Schizosaccharomyces
  • Schizosaccharomyces pombe Proteins
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Spliceosomes