Saccharomyces cerevisiae NineTeen complex (NTC)-associated factor Bud31/Ycr063w assembles on precatalytic spliceosomes and improves first and second step pre-mRNA splicing efficiency

Abstract

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a ∼25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.

FIGURE 1.

Bud31 is required for cell viability at 37 °C and for in vitro splicing at high temperatures. A, growth profile of serially diluted bud31 null and isogenic WT cultures at 23 and 37 °C. B, ³²P-labeled WT ACT1 transcripts spliced with WT (lanes 1–6) and bud31Δ extracts (lanes 7–12) at 23 and 37 °C. The substrate, reaction intermediates, and products are represented diagrammatically on the left. C, ratio of the first and second step catalyzed RNA species from triplicate experiments for the indicated reaction time points, normalized to the pre-mRNA in each case. The ratios shown are mean values with S.E.

FIGURE 2.

Bud31 stably associates with U5 and U6 snRNPs in splicing extracts. Bud31-TAP protein was immunoprecipitated from splicing extracts under increasing salt concentrations (NaCl conc., lanes 2–5). The co-immunoprecipitated U-rich snRNAs were detected by Northern blot of a denaturing urea PAGE gel. RNA from one-fifth of the extract used for immunoprecipitation was loaded as input. IgG-agarose pulldown from BY4743 extracts in 50 mm NaCl was loaded as control (C, lane 6).

FIGURE 3.

Bud31-polyoma middle T-antigen His-tagged protein is functional in vivo and associates with spliceosomes in vitro. A, growth profile of the bud31 transformed with pCHP425 plasmid expressing dual tagged Bud31 or after transformation with the empty vector at 23 or 37 °C. B, schematic representation of spliceosome assembly and the conditions that stall at B^act, B, and E complexes. UsnRNA, U-rich snRNA. C, in vitro reactions at 23 °C with 2 mm ATP (lanes 1–3), with 2 mm ATP and 5 mm EDTA to enrich B^act complex (lanes 4–6), with 0.05 mm ATP to obtain B complex (lanes 7–9), and with no ATP to arrest at E complex (lanes 10–12). The substrate RNA species in anti-polyoma-based pulldown from Bud31-associated spliceosomes is indicated in lanes marked as Ip. The nonspecific substrate association with beads is shown in lanes marked as B. RNA in one-fourth of the total reaction was loaded as input (I). D, the pre-mRNA in each lane was quantified and normalized to the input pre-mRNA, and the data from 3–5 experiments were plotted with error bars indicating S.E..

FIGURE 4.

Affinity-purified B and B^act spliceosomes analyzed for their snRNA content. A and B, splicing reactions performed with biotinylated mutant C303/C305 actin pre-mRNAs (A) or control nonbiotinylated pre-mRNAs (B). Reactions with 0.05 mm ATP and with 2 mm ATP supplemented with 5 mm EDTA were done with Bud31-TAP extracts. The substrate-associated spliceosomes were pulled down, and snRNA content was assessed by primer extension. Reactions with U1 and U4 snRNA primers were coupled (lanes 6, 8, 10, and 12 in panel A and lanes 1, 3, 5, and 7 in panel B), whereas those with U2, U5, and U6 snRNA primers were multiplexed (lanes 7, 9, 11, and 13 in panel A and lanes 2, 4, 6, and 8 in panel B). The reaction products were separated on denaturing PAGE. From each reaction condition, snRNAs in a direct load of an aliquot of the reactions (lanes 6, 7, 10, and 11 in panel A and lanes 1, 2, 5, and 6 in panel B) were compared with those in the affinity pulled down spliceosome (lanes 8, 9, 12, and 13 in panel A and lanes 3, 4, 7, and 8 in panel B). To accurately assign each U-rich snRNA, parallel primer extension reactions were done with total RNA from splicing extracts (panel A, lanes 1–5).

FIGURE 5.

Affinity-purified spliceosomes assembled on biotinylated and control pre-mRNAs probed for Bud31 and Prp21 proteins. Splicing reactions were performed with biotinylated mutant C303/C305 actin pre-mRNAs (lanes 3 and 5) or control nonbiotinylated pre-mRNAs (lanes 2 and 4) and extracts containing Bud31-TAP (upper panel) or Prp21-TAP (lower panel). The reaction conditions, as shown in Fig. 4, were enriched for B (lanes 2 and 3) and B^act complexes (lanes 4 and 5) as indicated above each lane. The proteins present in affinity-purified spliceosomes were probed by Western analyses with anti-TAP antibodies. The input lane had a direct load of proteins in the splicing extract (lane 1).

FIGURE 6.

Bud31 does not have direct interaction with pre-mRNA. In vitro splicing reactions were performed with splicing extracts from Bud31-TAP strain and, as a control, the Cwc2-TAP strain. The reactions were assembled on biotinylated actin pre-mRNA truncated before the 3′ splice site. The spliceosomes were UV cross-linked at 250 nm and purified by streptavidin affinity purification. The presence of substrate-associated Bud31 and Cwc2 in the enriched A1 spliceosomes was assessed by Western blotting with anti-TAP antibodies.

FIGURE 7.

Bud31 mediates interaction of an ∼25-kDa protein to −3 residue in exon 1 of CYH2 pre-mRNA. Top, in vitro splicing reactions with control CYH2 pre-mRNA with no 4-thioU (lanes 1–4) and with a CYH2 pre-mRNA with 4-thioU at the −3 position of the 5′ exon (lanes 5–8). A single radiolabeled phosphodiester bond was introduced two residues downstream to the −3 position in both control and 4-thioU substrate. Using splicing extracts from WT and bud31 null strains, the reactions were performed for 15 minutes at 23 or 37 °C. All reactions were UV cross-linked at 365 nm at 4 °C and then treated with RNase T1. The cross-linked proteins were separated on a SDS-PAGE gel and visualized by autoradiography. Bottom, the quantitation of the levels of the 25-kDa protein normalized to the 37-kDa protein detected in various reactions.