Project description:There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 is an RNA-stimulated ATPase required for pre-spliceosome formation in yeast. We demonstrate through in vivo RNA labelling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at serine 5 of the largest subunit’s heptad repeat accumulates over introns, and that this defect requires the U2 snRNP-associated Cus2p. A similar accumulation of polymerase was observed when pre-spliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with pre-spliceosome formation during co-transcriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription. Examining the Pol II profile in MT strain and WT strain
Project description:There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 is an RNA-stimulated ATPase required for pre-spliceosome formation in yeast. We demonstrate through in vivo RNA labelling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at serine 5 of the largest subunit’s heptad repeat accumulates over introns, and that this defect requires the U2 snRNP-associated Cus2p. A similar accumulation of polymerase was observed when pre-spliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with pre-spliceosome formation during co-transcriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription.
Project description:Pre-mRNA splicing is vital for the proper function and regulation of eukaryotic gene expression. Saccharomyces cerevisiae has been used as a model organism for studies of RNA splicing because of the striking conservation of the spliceosome and its catalytic activity. Nonetheless, there are relatively few annotated alternative splice forms, particularly when compared to higher eukaryotes. Here, we describe a method to combine large scale RNA sequencing data to accurately discover novel splice isoforms in Saccharomyces cerevisiae. Using our method, we find extensive evidence for novel splicing of annotated intron-containing genes as well as genes without previously annotated introns and splicing of transcripts that are antisense to annotated genes. By incorporating several mutant strains at varied temperatures, we find conditions which lead to differences in alternative splice form usage. Despite this, every class and category of alternative splicing we find in our datasets is found, often at lower frequency, in wildtype cells under normal growth conditions. Together, these findings show that there is widespread splicing in Saccharomyces cerevisiae.
Project description:Splicing regulatory networks are essential components of eukaryotic gene expression programs, yet little is known about how they are integrated with transcriptional regulatory networks into coherent gene expression programs. Here we define the MER1 splicing regulatory network and examine its role in the gene expression program during meiosis in budding yeast. Mer1p splicing factor promotes splicing of just four pre-mRNAs. All four Mer1p-responsive genes also require Nam8p for splicing activation by Mer1p, however other genes require Nam8p but not Mer1p, exposing an overlapping meiotic splicing network controlled by Nam8p. MER1 mRNA and three of the four Mer1p substrate pre-mRNAs are induced by the transcriptional regulator Ume6p. This unusual arrangement delays expression of Mer1p-responsive genes relative to other genes under Ume6p control. Products of Mer1p-responsive genes are required for initiating and completing recombination, and for activation of Ndt80p, the transcriptional network that controls subsequent steps in the program. Thus the MER1 splicing regulatory network mediates the dependent relationship between the UME6 and NDT80 transcriptional regulatory networks in the meiotic gene expression program. This work reveals how splicing regulatory networks can be interlaced with transcriptional regulatory networks in eukaryotic gene expression programs. This SuperSeries is composed of the SubSeries listed below.
Project description:In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre-messenger RNA (pre-mRNA). Recent studies show the process of RNA-synthesis and RNA-processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited into chromatin by the SWR1-complex, is found near the 5’ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP, as H2A.Z shows extensive genetic interactions with U2 snRNP associated proteins, and RNA-seq reveals introns with non-consensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z’s role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled and unspliced RNAs are released. Together these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.
Project description:Pre-mRNA splicing is a highly regulated process catalyzing intron excision by spliceosome. Spliceosome activation is a major control step requiring dramatic protein and RNA rearrangements leading to a catalytically active complex. Prior research has linked hyperphosphorylation of SF3B1, a subunit of U2 snRNP, with spliceosome activation and catalytically active spliceosome, rendering a relevant kinase a key player for pre-mRNA splicing. Here we use OTS964, the first potent inhibitor of cyclin-dependent kinase 11 (CDK11), to show rapid and selective dephosphorylation of SF3B1 on threonines required for spliceosome activation. CDK11 associates with SF3B1 and its inhibition causes massive intron retention, block in precatalytic spliceosome complex B to activated spliceosome complex Bact transition and accumulation of non-functional spliceosomes on pre-mRNA and chromatin. These studies reveal crucial regulatory role of CDK11 in human pre-mRNA splicing and define the compound OTS964 as a quality chemical biology probe for CDK11.
Project description:Pre-mRNA splicing is a highly regulated process catalyzing intron excision by spliceosome. Spliceosome activation is a major control step requiring dramatic protein and RNA rearrangements leading to a catalytically active complex. Prior research has linked hyperphosphorylation of SF3B1, a subunit of U2 snRNP, with spliceosome activation and catalytically active spliceosome, rendering a relevant kinase a key player for pre-mRNA splicing. Here we use OTS964, the first potent inhibitor of cyclin-dependent kinase 11 (CDK11), to show rapid and selective dephosphorylation of SF3B1 on threonines required for spliceosome activation. CDK11 associates with SF3B1 and its inhibition causes massive intron retention, block in precatalytic spliceosome complex B to activated spliceosome complex Bact transition and accumulation of non-functional spliceosomes on pre-mRNA and chromatin. These studies reveal crucial regulatory role of CDK11 in human pre-mRNA splicing and define the compound OTS964 as a quality chemical biology probe for CDK11.