Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs

Abstract

Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

FIG. 1.

Characterization of the TAP complexes. (A) Protein lysates from the Cdc5p-TAP-producing strain (upper panel) or the Cdc5p-TAP complex following its purification (lower panel) were resolved on a 10 to 30% sucrose gradient, and fractions were collected from the bottom (fraction 1). These were resolved by SDS-PAGE and immunoblotted with anti-Cdc5p serum. The migrations of FAS (40S), thyroglobulin (19S), and catalase (11.3S) collected from parallel gradients are indicated. (B) Silver-stained gel of fractions of the 10 to 30% sucrose gradient resolving the Cdc5p-TAP complex. Asterisks indicate fractions containing multiple peptides. Brackets labeled with the letter K indicate trace amounts of unavoidable human keratin contamination. (C) Silver-stained gels of a portion of each TAP complex. The protein compositions of mock purifications from wild-type S. pombe (KGY246) and wild-type S. cerevisiae (YPH09) were also examined by silver staining. (D) Electron microscopic analysis of the Cdc5p-TAP complex negatively stained with 0.75% uranyl formate. Shown is a gallery of selected particles representing different views of the complex. (E) Cdc5p-TAP associates with U2, U5, and U6 snRNAs. RNA was isolated from Cdc5p-TAP and from an anti-snRNA cap (antitrimethylguanosine [m₃G]) immunoprecipitation from wild-type cells. Blots were probed with ³²P-labeled oligonucleotides complementary to the S. pombe U1, U2, U4, U5, and U6 snRNAs. In the cases of the U2, U5, and U6 snRNAs for the TAP samples, exposures were 1/24 of the others. (F) Prp19p-TAP associates with U2, U5, and U6 snRNAs. RNA was isolated from Prp19p-TAP and from an anti-snRNA cap (anti-m3G) immunoprecipitation from wild-type cells. Blots were probed with ³²P-labeled oligonucleotides complementary to the S. cerevisiae U1, U2, U4, U5, and U6 snRNAs. In the cases of the U2, U5, and U6 snRNAs for the TAP samples, exposures were 1/12 of the others.

FIG. 2.

Validation of TAP/DALPC results. (A) S. pombe Cwf5p-myc, Cwf7p-HA, Cwf11p-myc, Cwf12p-HA, Cwf13p-HA, and Cwf17p-myc associate with Cdc5p in vivo. An anti-Cdc5 (left panel) and an anti-myc (right panel) immunoblot of immunoprecipitates (IP) from cwf5-myc, cwf7-HA, cwf11-myc, cwf12-HA, cwf13-HA, and cwf17-myc strains are shown. Immunoprecipitations were performed with preimmune sera (PI), anti-Cdc5p immune sera (I), anti-myc antibodies (myc), or anti-HA antibodies. (B) S. cerevisiae Cwc14p-myc and Cwc15p-myc associate with Cef1p in vivo. An anti-Cef1p (left panel) and an anti-myc (right panel) immunoblot of immunoprecipitates from cwc14-myc and cwc15-myc strains are shown. Immunoprecipitations were performed with preimmune sera (PI), anti-Cef1p immune sera (I), or anti-myc antibodies (myc). (C) S. pombe Lsm8p-TAP and Cdc5p coimmunoprecipitate. Shown is an anti-Cdc5p immunobolot of IgG pulldowns from wild-type (lane 1), arp2-TAP (lane 2), and lsm8-TAP (lane 3) strains.

FIG. 3.

Characterization of S. pombe Cwf11p. (A) The ClustalW 1.6 program (22, 67) was used to align Cwf11-related proteins from H. sapiens (Hs) (O060306), D. melanogaster (Dm) (Q9VGG7), C. elegans (Ce) (Q9U1Q7), A. thaliana (At) (Q9ZVJ8), and S. pombe (Sp) (O94508). Residues found to be identical or similar to those of human CWF11 are highlighted on a solid background or shaded, respectively. (B) cdc5-120 and cwf11::ura4⁺ cells show a reduced restrictive temperature. Wild-type, cdc5-120, cwf11::ura4, and cdc5-120 cwf11::ura4 mutant cells were streaked onto agar medium at 25°C. (C) Tetrads grown at 25°C from the cwf11HA/cwf11⁺ cdc5-120/cdc5⁺ diploid. Only very small colonies grew on plates containing G418, and these were temperature sensitive, indicating that they were double mutants.

FIG. 4.

Characterization of S. cerevisiae Cwc15p. (A) The ClustalW 1.6 program (22, 67) was used to align Cwc15-related proteins from H. sapiens (Hs) (Q9UI29), D. melanogaster (Dm) (Q9V3B6), C. elegans (Ce) (O45766), A. thaliana (At) (Q9LK52), S. pombe (Sp) (Cwf15p) (O74817), and S. cerevisiae (Sc) (Cwc15p) (Q03772). Residues found to be identical or similar to those of human CWF15 are highlighted on a solid background or shaded, respectively. (B) prp19-1 and cwc15::kan^r strains are synthetically lethal. A CEN URA3-marked plasmid carrying the PRP19 gene was introduced into a MATa/α prp19-1/PRP19 cwc15::kan^r heterozygote prior to sporulation. A prp19-1 cwc15::kan^r double-mutant strain expressing PRP19 from a URA3-based plasmid was isolated, struck to synthetic complete medium containing dextrose (SD) without Ura (SD − Ura) (left panel) or SD with Ura plus 5-fluoroorotic acid (SD + 5-FOA) (right panel), and incubated for 3 days at 25°C.

FIG. 4.

Characterization of S. cerevisiae Cwc15p. (A) The ClustalW 1.6 program (22, 67) was used to align Cwc15-related proteins from H. sapiens (Hs) (Q9UI29), D. melanogaster (Dm) (Q9V3B6), C. elegans (Ce) (O45766), A. thaliana (At) (Q9LK52), S. pombe (Sp) (Cwf15p) (O74817), and S. cerevisiae (Sc) (Cwc15p) (Q03772). Residues found to be identical or similar to those of human CWF15 are highlighted on a solid background or shaded, respectively. (B) prp19-1 and cwc15::kan^r strains are synthetically lethal. A CEN URA3-marked plasmid carrying the PRP19 gene was introduced into a MATa/α prp19-1/PRP19 cwc15::kan^r heterozygote prior to sporulation. A prp19-1 cwc15::kan^r double-mutant strain expressing PRP19 from a URA3-based plasmid was isolated, struck to synthetic complete medium containing dextrose (SD) without Ura (SD − Ura) (left panel) or SD with Ura plus 5-fluoroorotic acid (SD + 5-FOA) (right panel), and incubated for 3 days at 25°C.

FIG. 5.

Characterization of S. pombe Cwf7p. (A) The ClustalW 1.6 program (22, 67) was used to align Cwf7p-related proteins from H. sapiens (Hs) (O75934), D. melanogaster (Dm) (Q9VAY6), C. elegans (Ce) (Q22417), and S. pombe (Sp) (Q9USV3). Residues found to be identical or similar to those of human CWF7 are highlighted on a solid background or shaded, respectively. (B) cwf7⁺ is required for pre-mRNA splicing. RNAs were prepared from cwf7::ura4⁺ spores after germination, prp2-1 cells grown at 25°C or shifted to 36°C for 2 h, and wild-type cells and were then hybridized to oligonucleotides complementary to the tfIId or his3 mRNAs. PC, precursor; M, mature.

FIG. 5.

Characterization of S. pombe Cwf7p. (A) The ClustalW 1.6 program (22, 67) was used to align Cwf7p-related proteins from H. sapiens (Hs) (O75934), D. melanogaster (Dm) (Q9VAY6), C. elegans (Ce) (Q22417), and S. pombe (Sp) (Q9USV3). Residues found to be identical or similar to those of human CWF7 are highlighted on a solid background or shaded, respectively. (B) cwf7⁺ is required for pre-mRNA splicing. RNAs were prepared from cwf7::ura4⁺ spores after germination, prp2-1 cells grown at 25°C or shifted to 36°C for 2 h, and wild-type cells and were then hybridized to oligonucleotides complementary to the tfIId or his3 mRNAs. PC, precursor; M, mature.

FIG. 6.

Cdc5p remains in a large complex in pre-mRNA splicing mutants. (A) Lysates from prp1-1 cdc5-TAP, prp2-1 cdc5-TAP, prp4-1 cdc5-TAP, and prp12-1 cdc5-TAP strains after 3 h at 36°C, probed with anti-Cdc5p antibodies. Migration of FAS (40S), thyroglobulin (19S), and catalase (11.3S) collected from parallel gradients is indicated. (B) RT-PCR analysis of cdc2⁺ RNA isolated from wild-type (lane 2), prp1-1 cdc5-TAP (lane 3), prp2-1 cdc5-TAP (lane 4), prp4-1 cdc5-TAP (lane 5), and prp12-1 cdc5-TAP (lane 6) strains after 2 h at 36°C. Lane 1 represents a mock RT-PCR that lacks RNA but includes all other components of the reaction. PC, precursor; M, mature.