Myb-related fission yeast cdc5p is a component of a 40S snRNP-containing complex and is essential for pre-mRNA splicing.

The Schizosaccharomyces pombe cdc5+ gene was identified in the original screen for fission yeast mutants defective for cell cycle progression (51). At the restrictive temperature, cells harboring the temperature-sensitive cdc5-120 mutation become arrested with a 2N content of DNA in the G2 phase of the cycle (51, 56). The cloning and initial characterization of cdc5+ showed that its function is essential for viability and that its predicted protein product shares significant homology to the DNA binding domain of the vertebrate proto-oncoprotein c-Myb (56). The DNA binding domain of c-Myb consists of three imperfect repeats that contain evenly spaced hydrophobic residues, typically tryptophan but in some cases phenylalanine, isoleucine, or tyrosine (36). These residues have been shown by nuclear magnetic resonance structural analysis to make up the backbone of the DNA binding motif (53, 54). cdc5p contains two Myb repeats and an additional repeat that is similar to a Myb repeat but lacks certain residues characteristic of a canonical Myb repeat (55). Sequence similarity to a family of known DNA binding proteins led us to suggest initially that cdc5p might be required for entry into mitosis via regulation of transcription (56). Since the initial characterization of cdc5+, work in other organisms and database searches have revealed proteins that are closely related to cdc5p. To date, apparent cdc5p orthologs have been identified in Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Xenopus laevis, and human (7, 21, 25, 55, 69). This family of proteins was shown to be functionally conserved since DNAs encoding full-length versions of the Drosophila and Arabidopsis proteins and a truncated version of the human protein will rescue the growth defect of the S. pombe temperature-sensitive mutant, cdc5-120 (25, 55). Further the S. cerevisiae ortholog, CEF1 (S. cerevisiae homolog of cdc5), is essential for mitotic progression; cells lacking Cef1p arrest as large budded cells with aberrant spindle morphologies (55). Other links to cell cycle progression for cdc5 family members were reports that mammalian cells overexpressing hCdc5 showed a shortened G2 and reduced cell size, and cells expressing a carboxy-terminally truncated version of hCdc5 slowed G2 progression (6). Although it is highly conserved, the exact biochemical function of cdc5p/Cef1p has not been elucidated. Evidence supporting a role for this protein in DNA binding has been described. The bacterially expressed Myb repeats of the Arabidopsis thaliana ortholog bound to the double-stranded DNA sequence CTCAGCG in vitro (25). Also, the human ortholog was shown to be a nuclear protein (7) and a chimeric molecule consisting of the carboxy terminus of human Cdc5p coupled to the DNA binding domain of GAL4 transactivated a reporter gene in COS-7 cells (6). To address the function of cdc5p, we have examined its biochemical properties in S. pombe. As expected for a potential DNA binding protein, cdc5p was found exclusively within the nucleus. Unexpectedly, we also found that all detectable cdc5p is contained within a discrete 40S complex. Because the function of this complex seemed likely to be intimately entwined with that of cdc5p, we purified the complex and have identified 10 of its subunits. All are previously undescribed S. pombe open reading frames. Nine of the 10 components have obvious orthologs in other organisms, many of which are involved in pre-mRNA splicing. However, the functions of several orthologs have not been described. cdc5p also copurifies with the U2, U5, and U6 snRNAs demonstrating an association with S. pombe snRNPS. Consistent with this association, cells lacking cdc5+ function are defective in pre-mRNA splicing. Together, these data raise the possibility that the cdc5p complex is a stable intermediate in the assembly or disassembly of the S. pombe spliceosome. We discuss potential mechanisms whereby defects in pre-mRNA splicing give rise to cell cycle blocks.