Splicing factor Cwc22 is required for the function of Prp2 and for the spliceosome to escape from a futile pathway

Abstract

Cwc22 was previously identified to associate with the pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex [NTC]) involved in spliceosome activation. We show here that Cwc22 is required for pre-mRNA splicing both in vivo and in vitro but is neither tightly associated with the NTC nor required for spliceosome activation. Cwc22 is associated with the spliceosome prior to catalytic steps and remains associated throughout the reaction. The stable association of Cwc22 with the spliceosome requires the presence of the NTC but is independent of Prp2. Although Cwc22 is not required for the recruitment of Prp2 to the spliceosome, it is essential for the function of Prp2 in promoting the release of the U2 components SF3a and SF3b. In the absence of Cwc22, Prp2 can bind to the spliceosome but is dissociated upon ATP hydrolysis without promoting the release of SF3a/b. Thus, Cwc22 represents a novel ATP-dependent step one factor besides Prp2 and Spp2 and has a distinct role from that of Spp2 in mediating the function of Prp2.

FIG. 1.

Cwc22 is not a component of the NTC but is essential for pre-mRNA splicing both in vivo and in vitro. (A) Extracts prepared from PRP19-HA (lane 1), CWC22-HA (lane 2), and nontagged (lane 3) strains were immunoprecipitated with anti-HA antibody, followed by Western blotting using antibodies against the HA epitope and components of the NTC. (B) Growth curves of GAL-CWC22 cells in yeast extract-peptone-dextrose (YPD) and yeast extract-peptone galactose (YPG). Cells were grown in galactose-containing synthetic minimum medium to mid-log phase and then shifted to YPD or YPG medium. Cells were collected at 0, 4, 8, 12, 16, 20, 24, 28, 32, and 36 h after the shift for measurements of the optical density at 600 nm (OD₆₀₀). (C) Total RNA extracted from collected cells was analyzed by primer extension using a U3 primer, R13. The prp2 mutant was grown at 37°C for 2 h before harvesting. Gal, galactose; Glu, glucose; WT, wild type. (D) Splicing was carried out in mock-treated (lanes 1 and 4) or Cwc22-depleted (lanes 2, 3, 5, and 6) extracts using wild-type (lanes 1 to 3) or ACAC (lanes 4 to 6) actin pre-mRNA with (lanes 3 and 6) or without (lanes 1, 2, 4, and 5) the addition of 100 ng recombinant Cwc22. M, mock; D, depletion.

FIG. 2.

Deletion analysis of Cwc22. (A) Protein sequence alignment of Cwc22 orthologues using MAFFT and GeneDoc. Putative Cwc22 orthologues were identified by using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (17). Conserved residues are shaded in black for 100%, in gray for 80%, and in light gray for 60% conservation. Abbreviations: Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Rn, Rattus norvegicus; Mm, Mus musculus; Hs, Homo sapiens. (B) CWC22 deletion clones in yeast strain YSCC228 were grown in galactose-containing synthetic minimum medium and spotted onto glucose- or galactose-based plates after serial dilutions. (C) An estimated 1.5 μg each of purified Cwc22 (lane 1) and the segment at residues 212 to 491 (lane 2) was analyzed on 10% SDS-PAGE gels stained by Coomassie blue. (D) Recombinant Cwc22 at 5, 10, or 20 ng (lanes 3 to 5, respectively) or the segment at residues 212 to 491 at 10, 20, or 50 ng (lanes 6 to 8, respectively) was added to Cwc22-depleted extracts (lane 2) for splicing assays. M, mock; dCwc22, Cwc22 depletion; *, nonspecific band.

FIG. 2.

Deletion analysis of Cwc22. (A) Protein sequence alignment of Cwc22 orthologues using MAFFT and GeneDoc. Putative Cwc22 orthologues were identified by using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (17). Conserved residues are shaded in black for 100%, in gray for 80%, and in light gray for 60% conservation. Abbreviations: Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Rn, Rattus norvegicus; Mm, Mus musculus; Hs, Homo sapiens. (B) CWC22 deletion clones in yeast strain YSCC228 were grown in galactose-containing synthetic minimum medium and spotted onto glucose- or galactose-based plates after serial dilutions. (C) An estimated 1.5 μg each of purified Cwc22 (lane 1) and the segment at residues 212 to 491 (lane 2) was analyzed on 10% SDS-PAGE gels stained by Coomassie blue. (D) Recombinant Cwc22 at 5, 10, or 20 ng (lanes 3 to 5, respectively) or the segment at residues 212 to 491 at 10, 20, or 50 ng (lanes 6 to 8, respectively) was added to Cwc22-depleted extracts (lane 2) for splicing assays. M, mock; dCwc22, Cwc22 depletion; *, nonspecific band.

FIG. 3.

Cwc22 is associated with the spliceosome prior to the catalytic step and retained until completion of the reaction. Splicing was carried out with CWC22-HA extracts in the presence of 0.1 mM (lanes 1 to 4), 0.5 mM (lanes 5 to 8), or 2 mM (lanes 9 to 12) ATP, and the reaction mixtures were precipitated with anti-Ntc20 or anti-HA antibody. RXN, 1/10 of the reaction mixture used for immunoprecipitation; PAS, protein A-Sepharose.

FIG. 4.

Cwc22 is not required for NTC-mediated spliceosome activation. (A) The spliceosome formed with nonbiotinylated (lanes 1, 3, and 5) or biotinylated (lanes 2, 4, and 6) ACAC pre-mRNA in mock-treated (lanes 1 and 2), NTC-depleted (dNTC) (lanes 3 and 4), or Cwc22-depleted (lanes 5 and 6) extracts was isolated by precipitation with streptavidin-Sepharose, and the components were analyzed by Western blotting. (B) Splicing reactions were carried out with mock-depleted (lanes 2 to 4), NTC-depleted (lanes 5 to 7), Cwc22-depleted (lanes 8 to 10), or prp2-1 (lanes 11 to 13) extracts using biotinylated ACAC pre-mRNA as the substrate, and the spliceosome was precipitated with streptavidin-Sepharose. After washing off unbound materials, the pellet was separated into two fractions: one was used for total precipitate (lanes 2, 5, 8, and 11), and the other was added to splicing buffer and incubated for 20 min at room temperature. After separating the supernatant and pellet fractions, RNA was extracted and analyzed by Northern blotting. RNA, RNA from 2 μl of extracts; T, total precipitate; P, pellet; S, supernatant.

FIG. 5.

Cwc22 and Prp2 bind to the spliceosome independently of each other. Splicing was carried with ACAC pre-mRNA in these experiments. (A) Splicing was carried out with mock-treated (lane 1) or Cwc22-depleted (lanes 2 to 7) extracts without (lanes 2 to 6) or with (lane 7) the addition of recombinant Cwc22. Glucose (lanes 2 and 3) or ATP (lanes 4 and 5) was added to the reaction mixtures and incubated for 5 min. Cwc22 was then added, and the reaction mixture was incubated for 20 min. (B) Splicing was carried out with wild-type (lane 1), mock-treated (lanes 2 to 5), NTC-depleted (lanes 6 to 9), or Cwc22-depleted (lanes 10 to 13) extracts. After the addition of recombinant V5-tagged prp2_S378L, the reaction mixtures were precipitated with anti-Ntc20 or anti-V5 antibody. RXN, 1/5 of the reaction mixture used for immunoprecipitation; PAS, protein A-Sepharose. (C) Splicing was carried out with mock-treated (lanes 1 to 8) or Cwc22-depleted (lanes 9 to 16) Prp2-V5 extracts. Following the addition of glucose (lanes 1 to 4 and 9 to 12) or ATP (lanes 5 to 8 and 13 to 16) and incubation for 5 min, the reaction mixtures were precipitated with anti-Ntc20 or anti-V5 antibody. RXN, 1/10 of the reaction mixture used for immunoprecipitation; PAS, protein A-Sepharose. (D) Splicing was carried out with mock-treated (lanes 1 to 7) or Cwc22-depleted (lanes 8 to 14) Prp2-V5 extracts or with Cwc22-depleted extracts with the addition of the prp2_S378L-V5 protein (lanes 15 to 20). Following the addition of glucose and incubation for 5 min, the reaction mixtures were precipitated with anti-V5 antibody. The precipitates were reincubated in splicing buffer containing (lanes 2 to 4, 9 to 11, 17, and 18) or not containing (lanes 5 to 7, 12 to 14, 19, and 20) ATP, and supernatant and pellet fractions were separated. The released materials (lanes 22 and 26, as from lanes 4 and 11) were incubated for 20 min following the addition (lanes 24 and 28) or no addition (lanes 23 and 27) of micrococcal nuclease-treated extracts. R, 1/10 of the reaction mixture used for immunoprecipitation; T, total precipitates; P, pellet; S, supernatant; Ext^MN, micrococcal nuclease-treated extracts. (E) The spliceosome formed with biotinylated ACAC pre-mRNA in wild-type (lane 1) or Δprp2 extracts without (lane 2) or with (lane 3) the addition of the Prp2 protein was isolated by precipitation with streptavidin-Sepharose, and the components were analyzed by Western blotting.

FIG. 6.

Cwc22 is required for the function of Prp2. (A) Splicing was carried out with Cwc22-depleted extracts with ACAC pre-mRNA, and the reaction mixtures were precipitated with anti-Ntc20 antibody (lanes 3 to 11). Micrococcal nuclease-treated wild-type (lanes 4, 5, 8, and 9) or Δprp2 (lanes 6, 7, 10, and 11) extracts and wild-type (lanes 7 and 11), S378L (lanes 5 and 9), or no (lanes 4, 6, 8, and 10) Prp2 protein were added to the precipitated spliceosome and incubated in the presence (lanes 4 to 7) or absence (lanes 8 to 11) of ATP. The amounts of pre-mRNA and lariat intron-exon 2 were quantified by PhosphorImager analysis and, after conversion into molar amounts, plotted in a bar graph, with their sum as 100%. R, 1/10 of the reaction mixture; N, no antibody; WT, wild type; Δprp2, heat-treated prp2; m, mutant; Ext^MN, micrococcal nuclease-treated extracts. (B) Splicing was carried out in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of biotinylated ACAC pre-mRNA in mock-treated (lanes 1 and 2), Yju2-depleted (lanes 3 and 4), or Cwc22-depleted (lanes 5 and 6) Lea1-V5, Prp9-V5, or Hsh155-HA extracts. The spliceosomes were isolated by precipitation with streptavidin-Sepharose and analyzed by Western blotting using anti-V5, anti-HA, anti-Snu114, and anti-Ntc85 antibodies. Protein bands were quantified by using the UVP Biospectrum 600 imaging system and plotted in a bar graph, with each protein normalized to mock. dYju2, Yju2 depletion; dCwc22, Cwc22 depletion.

FIG. 7.

Schematic of the Cwc22 requirement to direct Prp2 into the productive pathway. After the activation of the spliceosome, Cwc22 and Prp2/Spp2 can bind to the spliceosome independently of each other. In the absence of Cwc22, Prp2 and Spp2 are dissociated from the spliceosome upon ATP hydrolysis with SF3a/b unreleased. In the presence of Cwc22, SF3a and SF3b are released with Prp2/Spp2 upon ATP hydrolysis. Cwc25 is then recruited to the spliceosome to promote the first catalytic reaction. Yju2 and HP-X are required for the first catalytic reaction after Prp2 action but can join the spliceosome at earlier steps, which are shown in gray to indicate their presence being functionally irrelevant. The question mark for HP-X indicates no experimental evidence for whether it can bind to the spliceosome prior to Cwc22 binding.