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.