Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bhaumik, Sukesh

Abstract

A characteristic feature of gene expression in eukaryotes is the addition of a 5' terminal 7-methylguanine "cap" to nascent pre-messenger RNA (mRNA) in the nucleus. It is the 5'capping process, which proves vital to creating a mature mRNA. The synthesis of an mRNA followed by its capping is a complex undertaking which requires a set of protein factors. The capped mRNA is then exclusively bound by a cap-binding complex (CBC). CBC shields mRNA from exonucleases as well as regulates downstream post-transcriptional events, translational initiation and nonsense mediated mRNA decay (NMD). Any misregulation during capping or in the binding of CBC can lead a number of diseases/disorders. Thus, the process and regulation of capping and CBC binding to mRNA are important fields to study the control of gene expression. Over the years, capping apparatus and CBC have been implicated in post-transcriptional regulation. However, it is not yet known whether CBC plays any role in controlling transcriptional initiation or elongation. Thus, the major research focus in my thesis had been to analyze the role of CBC and capping enzymes in regulation of transcriptional initiation and elongation. The results have revealed the role of CBC in stimulating the formation of pre-initiation complex (PIC) at the promoter in vivo via Mot1p (modifier of transcription). Subsequently, we have demonstrated the roles of CBC in transcription elongation, splicing and nuclear export of mRNA. Interestingly, we find that the capping enzyme, Cet1p, decreases promoter proximal accumulation of RNA polymerase II. These results support that Cet1p promotes the release of paused-RNA polymerase II to get engaged into elongating form for productive transcription. Such function of Cet1p appears to be mediated via the Facilitates chromatin transcription (FACT) complex. We find that FACT is targeted to the active gene by the N-terminal domain of Cet1p independently of its capping activity. In the absence of Cet1p, recruitment of FACT to the active gene is impaired, leading to paused-RNA polymerase II. Collectively, the results of my thesis work provide significant insight on the regulation of gene expression by CBC and capping enzyme, Cet1p.

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