Date of Award

12-1-2019

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bhaumik, Sukesh

Abstract

Eukaryotic gene expression is a highly synchronized cellular process whose nuclear phase is comprised of transcription, and mRNA processing and export. Transcription can be further comprised of transcription initiation, and elongation. Regulation of transcription initiation, transcription elongation, and mRNA processing and export are crucial for normal cellular function, since misregulation of these processes are associated with various diseases including cancer. Many factors or proteins are associated with these cellular processes which are modulated by different regulatory processes to maintain normal cellular function. Ubiquitin-proteasome system (UPS) is one of the recently studied regulatory processes. Over the years, ubiquitin and 26S proteasome have emerged as important regulatory factors in coordination of transcription and coupled mRNA export. However, the mechanisms as to how the ubiquitin and 26S proteasome regulate transcription and coupled mRNA export have not been clearly elucidated. Therefore, my dissertation has focused on understanding the role of UPS in these important cellular processes: transcription initiation, transcription elongation and mRNA export. The results have shown the non-proteolytic role of 19S RP of 26S proteasome in regulation of transcriptional initiation of SAGA and TFIID-dependent PHO84 gene. It was found that 19S RP facilitates both SAGA- and NuA4-TFIID-dependent transcriptional initiations of PHO84 via increased recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation in the presence and absence of an essential nutrient, Pi, in the growth media for transcriptional initiation, respectively. Next, our studies have uncovered the role of UPS in regulation of transcriptional elongation. It was found that E3 ubiquitin ligase, San1, mediated UPS regulation of transcription elongation factor, FACT is required for stimulating nucleosomal reassembly at the coding sequence of active genes for proper transcription elongation. We also found the interaction of FACT with another important transcription elongation factor, Paf1C via NTD (N-ter domain) of Cet1p (mRNA capping enzyme) to regulate transcription elongation.Subsequently, our results revealed a novel regulation of Paf1 component of Paf1C by UPS to regulate its abundance for proper cellular function. Transcription of genes could be blocked by DNA damage which can be repaired by transcription-coupled DNA repair (TCR) pathways. SUMOylation, another PTM (Post-translational modifications) like ubiquitination, is implicated in regulation of many DNA repair pathways including TCR, but it is not clearly understood how SUMOylation and associated enzymes are involved in regulation of such pathways. Here, we revealed the distinct role of SUMO ligases Siz1 and Siz2 in response to several DNA damaging agents such as UV, MMS (methyl methanesulfonate), HU (Hydroxyurea) and H2O2 (Hydrogen peroxide). Finally, we have extended our research works to understand the regulatory mechanisms of mRNA export by UPS. We found the interaction of TREX (Transcription/Export) component Sub2 with Mdm30 (F-box protein) for ubiquitination and proteasomal degradation of Sub2 in a transcription-dependent manner to regulate mRNA export. We also found the role CBC (Cap binding complex) in regulation of nuclear mRNA export. Collectively, the results of this study postulate a better understanding of regulation of transcription initiation, transcription elongation, and mRNA export by UPS.

Available for download on Wednesday, February 23, 2022

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