Acetylation by the Transcriptional Coactivator Gcn5 Plays a Novel Role in Co-Transcriptional Spliceosome Assembly

In the last several years, a number of studies have shown that spliceosome assembly and splicing catalysis can occur co-transcriptionally. However, it has been unclear which specific transcription factors play key roles in coupling splicing to transcription and the mechanisms through which they act. Here we report the discovery that Gcn5, which encodes the histone acetyltransferase (HAT) activity of the SAGA complex, has genetic interactions with the genes encoding the heterodimeric U2 snRNP proteins Msl1 and Lea1. These interactions are dependent upon the HAT activity of Gcn5, suggesting a functional relationship between Gcn5 HAT activity and Msl1/Lea1 function. To understand the relationship between Gcn5 and Msl1/Lea1, we carried out an analysis of Gcn5's role in co-transcriptional recruitment of Msl1 and Lea1 to pre-mRNA and found that Gcn5 HAT activity is required for co-transcriptional recruitment of the U2 snRNP (and subsequent snRNP) components to the branchpoint, while it is not required for U1 recruitment. Although previous studies suggest that transcription elongation can alter co-transcriptional pre-mRNA splicing, we do not observe evidence of defective transcription elongation for these genes in the absence of Gcn5, while Gcn5-dependent histone acetylation is enriched in the promoter regions. Unexpectedly, we also observe Msl1 enrichment in the promoter region for wild-type cells and cells lacking Gcn5, indicating that Msl1 recruitment during active transcription can occur independently of its association at the branchpoint region. These results demonstrate a novel role for acetylation by SAGA in co-transcriptional recruitment of the U2 snRNP and recognition of the intron branchpoint

Histone acetylation dynamics play a critical role in co- transcriptional spliceosome assembly and spliceosomal rearrangements

In the last several years, a number of studies have shown that spliceosome assembly and splicing catalysis can occur co-transcriptionally. However, it has been unclear which specific transcription factors play key roles in coupling splicing to transcription and the mechanisms through which they act. Here we report the discovery that Gcn5, which encodes the histone acetyltransferase (HAT) activity of the SAGA complex, has HAT-dependent genetic interactions with the genes encoding the heterodimeric U2 snRNP proteins Msl1 and Lea1, suggesting a functional relationship between Gcn5 HAT activity and Msl1/Lea1 function. To understand this relationship, we carried out an analysis of Gcn5's role in co-transcriptional recruitment of Msl1 and Lea1 to pre-mRNA and find that Gcn5 HAT activity is required for co-transcriptional recruitment of the U2 snRNP (and subsequent snRNP) components to the branchpoint. Although previous studies suggested that transcription elongation can alter co- transcriptional pre-mRNA splicing, we do not observe evidence of defective transcription elongation for these genes in the absence of Gcn5, while Gcn5-dependent histone acetylation is enriched in the promoter regions. While all these data suggest a role for histone acetylation in co- transcriptional spliceosome assembly. A closer examination of the functional interactions between histone mutants and the U2 snRNP and the effects of histone mutants and histone deacetylation on spliceosome assembly provide convincing evidence of the functional coordination of histone deaceylation and splicing. Mutations in histone residues targeted by Gcn5 show genetic interactions with the U2 snRNP and splicing defects that mirror GCN5 deletion. Furthermore, not only is Gcn5 associated throughout intron-containing genes, but deletion of multiple HDACs reveals peaks in acetylation in these regions, and this results in defects in spliceosome assembly. Finally, we present data that support a model whereby the Gcn5-dependent U2 snRNP recruitment facilitates HDAC recruitment, suggesting that splicing factors can, in fact, affect histone acetylation. These studies show that co-transcriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby spliceosome assembly is tightly coupled to histone modificatio

Acetylation by the transcriptional coactivator Gcn5 plays a novel role in co-transcriptional spliceosome assembly.

In the last several years, a number of studies have shown that spliceosome assembly and splicing catalysis can occur co-transcriptionally. However, it has been unclear which specific transcription factors play key roles in coupling splicing to transcription and the mechanisms through which they act. Here we report the discovery that Gcn5, which encodes the histone acetyltransferase (HAT) activity of the SAGA complex, has genetic interactions with the genes encoding the heterodimeric U2 snRNP proteins Msl1 and Lea1. These interactions are dependent upon the HAT activity of Gcn5, suggesting a functional relationship between Gcn5 HAT activity and Msl1/Lea1 function. To understand the relationship between Gcn5 and Msl1/Lea1, we carried out an analysis of Gcn5's role in co-transcriptional recruitment of Msl1 and Lea1 to pre-mRNA and found that Gcn5 HAT activity is required for co-transcriptional recruitment of the U2 snRNP (and subsequent snRNP) components to the branchpoint, while it is not required for U1 recruitment. Although previous studies suggest that transcription elongation can alter co-transcriptional pre-mRNA splicing, we do not observe evidence of defective transcription elongation for these genes in the absence of Gcn5, while Gcn5-dependent histone acetylation is enriched in the promoter regions. Unexpectedly, we also observe Msl1 enrichment in the promoter region for wild-type cells and cells lacking Gcn5, indicating that Msl1 recruitment during active transcription can occur independently of its association at the branchpoint region. These results demonstrate a novel role for acetylation by SAGA in co-transcriptional recruitment of the U2 snRNP and recognition of the intron branchpoint

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

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

Intracellular expression of <i>phhA</i> and growth of <i>L. pneumophila</i> in U937 cell macrophages.

<p>Intracellular expression of <i>phhA</i> and <i>letA</i> transcripts in macrophages, which were infected with wild-type (WT) 130b for 24 h, 48 and 72 h and then RT-PCR was done using primers that amplify the specific transcripts. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate reverse transcriptase (- RT). The results are representative of two independent experiments.</p