Mol Cell Biol. 1999 Aug;19(8):5352-62.
Myb-related fission yeast cdc5p is a component of a 40S snRNP-containing complex and is essential for pre-mRNA splicing.
McDonald WH1, Ohi R, Smelkova N, Frendewey D, Gould KL.
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Myb-related cdc5p is required for G(2)/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiae splicing factor Prp19p. cwf1(+) is allelic to the prp5(+) gene defined by the S. pombe splicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 and prp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lacking cdc5(+) function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.
Mol Cell Biol. 1999 Aug;19(8):5352-5362.
cdc5p is a component of a 40S complex. Shown is an immunoblot of fractions collected from 10 to 30% glycerol gradients which had been centrifuged for 15 h at 28,000 rpm. Strains from which lysates were prepared were as follows: 972 h− (wild type), cdc10-V50 block (G1 block), hydroxyurea block (HU) (S-phase block), cdc25-22 block (G2 block), nuc2-663 block (M-phase block), or cdc5-120 block. Blots were probed with anti-cdc5p. The migrations of FAS (40S), thyroglobulin (19S), and catalase (11.3S) collected from parallel gradients are indicated.
S. pombe cwf1p, cwf2p, cwf3p, cwf4p, and cwf10p associate with the cdc5p complex in vivo. (A) Immunoblots on fractions from 10 to 30% sucrose gradients probed with anti-cdc5 (cdc5p), anti-myc epitope, 9E10 (cwf3p-myc, cwf4p-myc, cwf1p-myc, cwf2p-myc, and cwf10p-myc, or anti-FAS (FAS-40S marker, FASp). The small asterisk on the right side of the panel indicates an apparent amino-terminal truncation of cwf1p-myc. The migration of thyroglobulin (19S) and catalase (11.3S) collected from parallel gradients is indicated. (B) Endogenously myc-tagged versions of cwf1p, cwf2p, cwf3p, cwf4p, and cwf10p coimmunoprecipitate with cdc5p in vivo. An anti-myc immunoblot of immunoprecipitates from wild-type (246), cwf3myc, cwf4myc, cwf1myc, cwf2myc, or cwf10myc strains is shown. Immunoprecipitations were performed with anti-cdc5p immune sera (I), preimmune sera (PI), or anti-myc antibodies (9E10, myc). Arrows indicate full-length tagged proteins. The small asterisk indicates an apparent amino-terminal truncation of cwf1p-myc. (C) Anti-cdc5p immunoblot of immunoprecipitates from cwf3myc, cwf4myc, cwf1myc, or cwf10myc strains with 9E10. The arrow indicates endogenous cdc5p. (D) Autoradiograph of immunoprecipitates from strains metabolically labeled with Tran[35S]-label as follows: wild-type, cdc5HA, cwf3HA, cwf4HA, and cwf1HA. The arrows indicate HA-tagged proteins in each respective strain. The circles indicate the untagged versions of the respective proteins. (E) cwf1+, cwf2+, cwf3+, and cwf4+ are essential genes. Shown are results for representative phenotypes of cwf::KanR spores.
cdc5-120 and prp5-1 show a reduced restrictive temperature. The wild type and prp5-1, cdc5-120, and cdc5-120 prp5-1 mutants were streaked onto agar medium at 25, 29, 32, and 36°C. The double-mutant strain was capable of growth at 25°C but not at 29°C. Each single mutant was capable of growth at both 29 and 32°C.
cdc5p associates with U2, U5, and U6 snRNAs. RNA was isolated from anti-m3G cap, anti-myc, or anti-HA immunoprecipitations from the wild-type, cdc5HA, or cdc5myc strain and subsequently probed with 32P-labeled oligonucleotides complementary to the U1, U2, U4, U5, or U6 snRNA.
cdc5+ function is required for pre-mRNA splicing. RNA was prepared from KGY1490 cells grown in the presence of thiamine for 0, 4, 8, or 12 h, prp2-1 cells grown at 37°C for 0, 2, or 4 h, or wild-type (wt) cells and then hybridized to oligonucleotides complementary to the U6 intron, mature U6 RNA, and both intron and exon sequences within the tf2d gene.