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Access Type
WSU Access
Date of Award
January 2024
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Biological Sciences
First Advisor
Athar Ansari
Abstract
Gene regulation in eukaryotes relies on complex, multi-layered processes that govern the precise control of transcription, the first step in gene expression. A growing body of evidence suggests that introns—non-coding regions within genes—play an integral role in regulating transcriptional activity beyond their traditional roles in RNA splicing. However, the exact mechanisms by which introns influence transcription remain poorly understood. This dissertation addresses these questions by examining the role of introns in transcriptional activation and the underlying mechanism that may facilitate this process in budding yeast.In preliminary investigations within our lab, the extent to which an intron can activate transcription of yeast genes was examined. It was observed that in the presence of an intron there is an augmentation in transcript levels of the cognate gene, underscoring the enhancement potential of introns on transcription. To further test the enhancement potential of an intron, I introduced the ACT1 intron into the promoter-proximal regions of four intronless yeast genes (INO1, MET16, CHA1, and GAL10) and examined their transcription levels under the influence of an intron versus a transcription activator. Remarkably, insertion of the intron restored transcription to levels comparable to those seen with active transcriptional activators, even in the absence of such activators. Transcription run-on assays further revealed that this intronic insertion could directly enhance transcription of nascent mRNA, indicating that the intron itself might promote transcriptional activation independently by facilitating the assembly of the preinitiation complex (PIC) at the promoter. Building upon these findings, I next investigated the broader regulatory mechanism in which splicing-competent introns enhance transcription. Based on studies from our lab demonstrating the formation of a gene loop—transcriptional structures formed by the interaction between promoter and terminator regions of a gene—in the presence of an activator or intron, I focused on the protein-protein interactions that might underscore loop-formation and subsequent enhanced transcription. Gene looping, previously shown to involve interactions between transcription factors such as TFIIB and termination complexes (CF1, CPF, and Rat1), was examined. Through quantitative proteomics of affinity-purified termination complexes, I discovered that termination factors not only interact with general transcription factors (i.e. TFIIB and TFIID) but also with splicing factors (Prp19, Prp43, Sub2, Snu114, Brr2, and Smb1), suggesting a collaborative role between transcription and splicing machinery in gene regulation. Notably, Prp19 emerged as a critical interactor with both transcription initiation and termination factors, prompting further analysis of its role in transcription. To elucidate the function of Prp19 in intron-mediated enhancement of transcription, I employed an auxin-inducible degron system to deplete Prp19, which resulted in a significant decrease in nascent transcription levels across both intron-containing and intron-less genes. Chromatin immunoprecipitation (ChIP) assays revealed that Prp19 associates with the promoter-proximal regions of these genes, supporting a novel role for Prp19 in the initiation of transcription. This effect appears mediated through the recruitment of the TFIID subunit TBP, linking Prp19 directly to transcription initiation and suggesting a broader regulatory function for splicing factors within transcription. Overall, this work demonstrates that introns can act independently to promote transcriptional activation, potentially by facilitating PIC assembly, and identifies Prp19 as a novel regulatory component in transcription initiation. These findings advance our understanding of how introns and associated protein complexes coordinate to regulate gene expression, offering new insights into the noncanonical roles of splicing factors and introns in transcription.
Recommended Citation
Dwyer, Katherine Marie, "Splicing-Dependent Regulation Of Transcription: A Novel Role Of The Splicing Factor Prp19 In Rnapii-Transcribed Genes" (2024). Wayne State University Dissertations. 4155.
https://digitalcommons.wayne.edu/oa_dissertations/4155