Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae. Here we show crucial roles for the chromatin remodeling complex SWI/SNF in splicing regulation in response to environmental changes. Nutrient-dependent downregulation of Snf2, the ATPase subunit of SWI/SNF, regulates downregulation of ribosomal protein genes (RPGs). RPGs are intron-enriched, and are highly transcribed. We show that their downregulation causes spliceosome redistribution from this abundant class of intron-containing RNAs to transcripts containing non-canonical splice-signals, which otherwise have poor affinity for the spliceosome.

Meiosis in S. cerevisiae is a response to prolonged starvation, involving regulated transcription and splicing of meiosis-specific transcripts. Splicing of a subset of these relies upon the meiosis-specific splicing activator Mer1. We find that SWI/SNF affects meiotic splicing in multiple ways. First, meiosis-specific downregulation of Snf2 leads to RPG downregulation and spliceosome redistribution to Mer1-regulated transcripts. Secondly, Mer1 expression is SWI/SNF dependent—Snf2 is poised at the MER1 promoter, and timing of Snf2 downregulation in relation to acetylation states of both itself and its target genomic loci allows coordination between these mechanisms. Hence, the SWI/SNF complex directs regulated meiotic splicing in S. cerevisiae. Furthermore, Snf2 itself is subject to precise regulation in response to cellular needs via several novel modes of RNA processing and regulation, as well as control of protein acetylation and turnover. This multi-level coordinated regulation orchestrates activity and targets of splicing programs as a cellular adaptive strategy in response to environmental stresses.

We also report roles for the SWI/SNF complex in respiration, partially via splicing regulation. Nutrient-dependent decrease in Snf2 leads to increase in PTC7 splicing, due to RPG downregulation and spliceosome redistribution. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of Coenzyme Q6 (CoQ6) biosynthesis, a mitochondrial redox-active lipid essential for respiration, and increased PTC7 splicing increases CoQ6 levels. Contrastingly, the nonspliced PTC7 isoform encodes a protein repressing CoQ6 biosynthesis via as-yet-unknown mechanisms. These findings establish a novel role for SWI/SNF in the transition of yeast cells from fermentative to respiratory metabolism. Overall, the SWI/SNF complex regulates cellular stress responses by redirecting energy from translation to specialized splicing programs.

Because RNA-synthesis and RNA-processing are spatially and temporally coordinated, RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, the SWR-C complex deposits H2A.Z into chromatin near the beginning of protein-coding genes where it helps regulate transcription. In this dissertation we elucidate a role for H2A.Z in coordinating the processes of RNA transcription and pre-mRNA splicing.

H2A.Z is required for optimal splicing of intron-containing genes, particularly under suboptimal splicing conditions. H2A.Z genetically interacts with the, particularly with U2 snRNP complex, and is required for efficient spliceosome rearrangements. Loss of H2A.Z results in defective spliceosome rearrangements, particularly those involving the U2 snRNP. H2A.Z loss impairs transcription elongation, suggesting that spliceosome rearrangements are tied to the role of H2A.Z 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. 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.

Chromatin not only affects splicing locally but also globally. We demonstrate that a chromatin remodeler regulates respiration through modulation spliceosome availability. Nutrient-responsive decrease in the Snf2 chromatin remodeler leads to ribosomal protein gene (RPG) down-regulation. Because RPGs are intron-enriched and highly transcribed, this relieves competition for limiting spliceosomes and allows for increased splicing of weaker substrates, such as PTC7. The spliced PTC7 isoform encodes a mitochondrial protein that promotes Coenzyme Q6 biosynthesis during respiration. These findings establish a role for the SWI/SNF complex in yeast in transition to respiratory metabolism through global regulation of splicing.

Furthermore, we show that disruption of transcription elongation kinetics can effect splicing both locally and globally, resulting in multi-faceted changes in splicing outcomes. While loss of the elongation factor Dst1 exacerbates transcription and splicing defects in cells lacking H2A.Z, it can also improve splicing of many intron-containing genes. In the absence of both Dst1 and H2A.Z, RPGs are down-regulated, allowing for spliceosome redistribution to and increased splicing of weaker substrates.

Additionally, we show that H2A.Z is required for growth in alkaline pH and expression of phosphate-starvation response genes. However, we find that, when H2A.Z is lost, deletion of Swr1 suppresses alkaline sensitivity, relieves aberrant RNA polymerase II (RNAPII) phosphorylation, and restores appropriate phosphate-gene. Therefore, stress sensitivity and transcription misregulation may be due to deleterious effects of an incomplete SWR-C complex, demonstrating the need for precise control of chromatin remodeling activity in the coordination of gene expression processes.

Eukaryotic organisms have evolved complex gene regulatory networks to launch coordinated responses to external conditions and stimuli. Under environmental stress, such as nutrient depletion, these responses involve reallocation of cellular resources away from the products of growth and cell cycle stimulating genes and towards the products of stress-responsive genes. This occurs through several mechanisms, including changes in transcription, decreased ribosome biogenesis, altered translation initiation, and changes in mRNA features that modulate translation efficiency and transcript stability. Upon nutrient starvation in the yeast Saccharomyces cerevisiae, diploid cells undergo meiosis leading to sporulation. Previous studies show that the catalytic component of the Swi/Snf chromatin remodeling complex, Snf2, is responsible for shifting gene expression away from intron-rich ribosomal protein genes (RPGs) to enhance splicing of meiotic intron-containing genes (ICGs) during sporulation, and that a similar process occurs during the transition from growth to quiescence known as the diauxic shift in yeast. During both of these complex cell-state transitions, Snf2 protein levels change dramatically while SNF2 mRNA levels remain relatively stable.The aim of this study is to understand the mechanism by which Snf2 protein levels change in response to nutrients. Here I describe alternate transcription start sites (TSS) at the SNF2 gene locus under batch growth conditions, which produce SNF2 transcripts with distinct 5’ leaders affecting downstream translation propensity. Specifically, a long transcript isoform of SNF2 mRNA containing three upstream open reading frames (uORFs), is capable of inhibiting the translation of the downstream protein-coding ORF. I identified the previously unannotated TSS of the long SNF2 isoform and performed RNA analysis in mutant and wild-type cells under various conditions to demonstrate that the transcript isoforms undergo nutrient-responsive transcript isoform switching, which is affected by the transcriptional regulator Ume6. Parallel protein analysis via Western blotting shows that this regulation indeed affects Snf2 expression, and that there is an inverse relationship between expression of the long SNF2 isoform and Snf2 protein levels. In light of the conservation of Snf2-family proteins, investigating SNF2 regulation in response to environmental changes in S. cerevisiae may carry important implications for understanding the regulation of Swi/Snf chromatin remodeling activity in higher eukaryotes.

In the nucleus of the eukaryotic cell, spliceosome assembly and the subsequent catalytic steps of precursor RNA (pre-mRNA) splicing occur cotranscriptionally in the context of a dynamic chromatin environment. A key challenge in the field has been to understand the role that chromatin and, more specifically, histone modification plays in coordinating transcription and splicing. Despite previous work to decipher the coordination between these processes, a mechanistic understanding of the role that chromatin modification plays in spliceosome assembly has remained elusive.

Histone H3K36 trimethylation (H3K36me3) is a highly conserved histone mark found in the body of actively transcribed genes, making it a particularly attractive candidate for having a role in the regulation of pre-mRNA splicing. Although there have been reports that H3K36me3 influences alternative splicing, the underlying mechanisms have remained elusive. Moreover, despite the prevalence of this mark on actively transcribed genes across eukaryotes, there has not been a mechanistic dissection of its potential role in constitutive splicing. Here we describe how we have addressed these questions and uncovered a core role for H3K36 methylation (H3K36me) in splicing in Saccharomyces cerevisiae (S. cerevisiae).

RNA-seq analyses of yeast strains deleted of the histone methyltransferase SET2 or harboring a mutation at the residue lysine 36 revealed widespread and overlapping splicing changes. Through a combination of RNA-seq, ChIP-seq, and biochemical analyses, we determined that the H3K36me effects on splicing are not due defects in transcription, but rather to loss of binding of the chromodomain protein Eaf3. We further show that Eaf3 interacts with the splicing factor Prp45, the human ortholog of SKIP. Consistent with this, Eaf3 is required for the cotranscriptional recruitment of the Prp Nineteen Complex (NTC), which is required for spliceosome activation. Hence, loss of H3K36 methylation has a direct and crucial role in spliceosome assembly. We demonstrate a central role for a highly conserved histone modification in constitutive pre-mRNA splicing. We also show that the reciprocal is true—splicing affects chromatin state.

Although it is established that spliceosome assembly occurs cotranscriptionally, a question that remains is how the activities of the proteins that regulate the dynamic rearrangements of the spliceosome are modulated by transcription and the state of the chromatin. Prp43, an ATPase involved in spliceosome disassembly, has been implicated at numerous steps in the splicing cycle and is therefore an interesting target in the regulation of splicing. Our study aims to determine whether Prp43 plays a role in directing spliceosome rearrangements via chromatin interactions and how interactions between Prp43 and chromatin may affect splicing outcomes. We show that Prp43 associates with chromatin in an RNA-independent manner, but splicing-dependent manner. Furthermore, we observed that Prp43 interacts with H3K36me3, possibly for its role in spliceosome disassembly.

Pre-mRNA splicing is an essential step in expression of eukaryotic genes. In Saccharomyces cerevisiae, pre-mRNA splicing plays an important role in regulating proliferation and the cellular response to changes in nutrient availability. To study how defects in splicing affect cellular viability we performed a genetic screen to identify mutations that enhance growth of the temperature sensitive strain prp2-1, which contains a point mutation in the gene encoding the essential DEAH-box ATPase, Prp2. We identified intragenic mutations within PRP2 that enhance growth of the temperature sensitive allele prp2-1. A majority of these intragenic suppressor mutations were clustered around the ATP binding site of Prp2. The intragenic prp2 mutations identified by the screen also rescued the splicing and rRNA processing defects observed in prp2-1. This work provides insight into the mechanism by which the prp2-1, a commonly used allele to study the function of Prp2 and pre-mRNA splicing, affects RNA processing and cellular viability. Our genetic screen also identified mutations in the stress granule component, Pbp1, which improved the growth of prp2-1, but did not enhance splicing. We find that in addition to decreased production of functional mRNAs, inhibition of splicing in yeast leads to sequestration of Pbp1 in the nucleus during nutrient deprivation and prevention of Pbp1 from localizing to starvation-induced cytoplasmic stress granules. We find that removal of the putative RNA binding domain of Pbp1 prevents sequestration of Pbp1 in the nucleus and restores Pbp1 cytoplasmic condensate formation during nutrient deprivation. Together this work provides insight into the ways defects in pre-mRNA splicing affect cellular function and has implications for the study of human diseases that are impacted by mutations in splicing factors, such as various cancers and neurodegenerative diseases.