Dynamic histone acetylation is critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements

Abstract

Assembly of the spliceosome onto pre-mRNA is a dynamic process involving the ordered exchange of snRNPs to form the catalytically active spliceosome. These ordered rearrangements have recently been shown to occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template. We previously demonstrated that the histone acetyltransferase Gcn5 is required for U2 snRNP association with the branchpoint. Here we provide evidence that histone acetylation and deacetylation facilitate proper cotranscriptional association of spliceosomal snRNPs. As with GCN5, mutation or deletion of Gcn5-targeted histone H3 residues leads to synthetic lethality when combined with deletion of the genes encoding the U2 snRNP components Lea1 or Msl1. Gcn5 associates throughout intron-containing genes and, in the absence of the histone deacetylases Hos3 and Hos2, enhanced histone H3 acetylation is observed throughout the body of genes. Deletion of histone deacetylaces also results in persistent association of the U2 snRNP and a severe defect in the association of downstream factors. These studies show that cotranscriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby yeast spliceosome assembly is tightly coupled to histone modification.

Fig. 1.

Mutation of histone H3 residues combined with deletion of either MSL1 or LEA1 leads to severe synthetic growth defects. (A) Growth analysis of the double-mutant lea1Δ and histone H3 point mutants or truncation. Cells were grown at 30 °C in YPD medium until the desired OD₆₀₀ was obtained. Cells were spotted onto YPD plates as a 10-fold serial dilution and grown at 30 °C or 37 °C for 2 d. (B) Dilution series of the double mutant msl1Δ and histone H3 point mutants or truncation. Cells were treated as described in A. (C) Quantitative RT-PCR of DBP2 and ECM33 in the histone H3 truncation mutant histone H3Δ9–16 or gcn5Δ vs. wild-type. Data are represented as a fold-increase in the ratio of precursor (unspliced)/total DBP2 or ECM33 message relative to wild-type. Graph represents three independent experiments and error bars represent SEM.

Fig. 2.

Gcn5-dependent histone acetylation in the coding region of DBP2 is masked by histone deacetylation. (A) ChIP analysis of histone H3 K9/14 acetylation of DBP2 in wild-type and histone deacetylase mutants using an antibody that recognizes acetylated histones (06-599; Millipore). Data are represented as diacetylated histone H3 normalized to the total amount of histone H3 (Total H3). (B) ChIP analysis of diacetylation of histone H3 in wild-type and the HOS3 HOS2 double deletion strain for ECM33 (exons are shaded gray). Light gray bars represent wild-type and speckled bars represent the HDAC double mutant. Data are represented as diacetylated H3 normalized to total histone H3. Graphs represent the average of at least three independent experiments, ±SD

Fig. 3.

Deletion of HDACs Hos3 and Hos2 alters cotranscriptional recruitment of Msl1/Lea1. (A) Graphs depicting the occupancy of Lea1-HA and Msl1-HA at each region of DBP2 in the presence or absence of the HDACs, HOS3 and HOS2, relative to the nontranscribed control. Light gray bars depict the occupancy of Lea1 or Msl1 in the presence of HDACs. Dark gray bars represent Lea1-HA or Msl1-HA occupancy in the absence of HDACs. (B) Graph depicting the occupancy of Lea1-HA or Msl1-HA at each region of ECM33 in the presence and absence of HOS3 and HOS2 relative to the nontranscribed region. Data are represented as in A. Graphs represent the average of three independent experiments, ±SD. (C) Dilution series of the triple deletion mutants, lea1Δ hos3Δ hos2Δ and msl1Δ hos3Δ hos2Δ. Cells were grown at 30 °C in YPD liquid medium until the desired OD₆₀₀ was obtained. Cells were spotted onto YPD plates as a 10-fold serial dilution, and plates were incubated for 2 d at 30 °C, 3 d at 25 °C, and 5 d at 16 °C.

Fig. 4.

Deletion of the HDACs Hos3 and Hos2 affects spliceosome dynamics downstream of U2 snRNP recruitment. (A) Bar graph depicting the cotranscriptional recruitment of U5 snRNP (Sun114-HA) to DBP2 in the presence and absence of Hos3 and Hos2 (Left). Light gray bars represent the occupancy of Snu114-HA in a wild-type background and the dark gray bars represent Snu114-HA recruitment when HOS3 and HOS2 are deleted. Occupancy is measured as fold-accumulation over the nontranscribed control. Bar graph represents the recruitment of Prp19-HA to DBP2 in the presence and absence of Hos3 and Hos2 (Right). Light gray bars represent wild-type and dark gray bars represent the hos3Δ, hos2Δ double mutant strain. (B) Bar graph depicting the cotranscriptional recruitment of U5 snRNP (Snu114-HA) and Prp19-HA to ECM33 in the presence and absence of Hos3 and Hos2 (Left and Right, respectively). Data are represented as in A. Graphs represent the average of at least three independent experiments.

Fig. 5.

Histone acetylation and deacetylation play a role in cotranscriptional splicing. Model depicting the role of Gcn5 and HDACs in spliceosomal rearrangements. Gcn5-dependent histone acetylation may create binding sites for a factor that interacts directly or indirectly with U2 snRNP proteins (indicated by dotted lines), to facilitate their cotranscriptional association. Hos3- and Hos2-mediated deacetylation allows for the proper release of the U2 snRNP and assembly of the spliceosome. A testable prediction is that the U2 snRNP may affect recruitment of histone deacetylases.