Prp5-Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription

Abstract

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

Figure 1.

Reciprocal genetic interactions between transcription factor SPT8 and splicing factor PRP5. (A) Strategy for genetic screen for suppressors of the cold sensitive (cs) prp5-GAR allele. One spt8 mutant allele was obtained. (B) Deletion of SPT8 gene rescues the cs phenotype of prp5-GAR allele at 16°C. (C) At the permissive temperature (30°C), prp5 alleles (-SAG, -TAG and -GAG) rescue growth defects of spt8Δ allele on 6-AU.

Figure 2.

SPT3, another SAGA component, also genetically interacts with PRP5. (A) Illustration of the yeast SAGA complex. Components are classified into four groups (57), in which colored factors are tested in this study. (B) Deletion of SPT3 rescues the cs phenotype of the prp5-GAR allele, whereas deletion of other tested SAGA components cannot. (C) prp5 alleles (-TAG and -GAG) rescue growth defects of the spt3Δ allele on 6-AU.

Figure 3.

In the absence of SPT8, prp5 mutant alleles stimulate RNA polymerase II binding to an intron-containing gene. ChIP assays on intron-containing gene DBP2 and intronless gene PDR5 were performed using RNA Pol II antibody 8WG16 (A and A’), anti-HA agarose for the HA-tagged Spt8 strain (B) and for the HA-tagged Gcn5 strain (C), and anti-Flag agarose for the Flag-tagged Prp5 strains with WT SPT8 (D) or spt8Δ (E). Temperatures for yeast cultures and location of PCR primers are indicated. Bar graphs represent the co-immunoprecipitated signals and were normalized to input. All the data were analyzed from triplicates. Error bars represent standard deviation from the mean.

Figure 4.

ChIP-seq reveals that global RNA Pol II binding is mutually rescued by prp5-GAR and spt8Δ. (A) Inhibited splicing by the prp5-GAR allele is restored by SPT8 deletion. RT-PCRs were performed and the inhibited splicing ratios were semi-quantitated by signals of pre-mRNA / (pre-mRNA + mRNA) from triplicates. Analyses of genome-wide RNA Pol II binding on yeast genes in the presence of prp5-GAR, spt8Δ, and the double-mutant alleles at 30°C (B) and 16°C (B'). Intronless and intron-containing genes were separately analyzed, and genes were grouped into increased, no change and decreased RNA Pol II binding according to the total mapped reads that are normalized by input. Individual analyses of three genes with RNA Pol II pausing in the intronic regions suggested the restored pausing in the double-mutant strain (C).

Figure 5.

Prp5p physically interacts with Spt8p, but not Spt3p, in vivo and in vitro. (A) Spt8p interacts with Prp5p in cell lysate. In the cell lysate from a double-tagged strain, Spt8p-HA pulls down FLAG-Prp5p. Single-tagged strains are used as controls. (B) Prp5p directly interacts with Spt8p, but not Spt3p. Direct in vitro protein-protein interactions were tested using purified recombinant proteins and Ni-NTA agarose. Spt8p and Spt3p were GST-tagged, and Prp5p was fused to a 6xHis tag. No-bait pulldowns (i.e. beads alone) were used as controls. (C) The N-terminus of Prp5p is required for Spt8p–Prp5p interaction. Upper, schematic representation of the full-length and truncated Prp5p proteins. Lower, in vitro protein-protein interactions were tested as in panel B. (D) CUS2 is required for maintaining the genetic interaction between PRP5 and SPT8. cus2Δ abrogates the ability of all tested prp5 alleles to rescue the growth defects of spt8Δ on 6-AU medium at 16°C and 30°C.

Figure 6.

Deletion of the TBP-binding module components of SAGA complex, SPT8 or SPT3, reverses PRP5-dependent proofreading at branch site region. (A) Schematic of the ACT1-CUP1 reporter used for copper tolerance assay to monitor the efficiency of in vivo splicing. Mutant sites used in this study are indicated in red. (B) Deletion of SPT8 reverses the improvement of splicing activity of suboptimal branch site region substrates by prp5 alleles (SAT mutants). Growth of each strain on three representative copper concentrations was selected to illustrate altered splicing activities. (C) Deletion of SPT3, but not other transcription factors, also reverses proofreading at the branch site region by PRP5.

Figure 7.

Coupling between transcription and splicing mediated by Spt8p/Spt3p and Prp5p. (A) Schematic depicting recruitment of Prp5p to the promoter region by the TBP-binding module of SAGA (Spt8p and Spt3p) and subsequent modulation of splicing proofreading at the BS region by Prp5p. This recruitment relies on direct interaction between Prp5p and Spt8p. (B) Deletion of SPT8 results in altered transcription of a subset of intronless and intron-containing genes, most are down-regulated. Loss of SPT8/SPT3 increases splicing proofreading at the BS region. (C) The prp5 mutant allele affects transcription of a subset of intronless and intron-containing genes and inhibits pre-mRNA splicing. (D) The double mutant, prp5 allele and deletion of SPT8, restores both affected transcription and inhibited pre-mRNA splicing. Cus2p plays a critical role in this coupling, deletion of CUS2 abrogates the coupling between transcription and splicing. Lack of Spt8p/Spt3p results in increased splicing proofreading on suboptimal BS substrates. For clarity, intronless genes are not depicted.