Open Access Dissertation
Date of Award
It is now quite evident that the introns, which are removed from the primary transcript by the process of splicing, are involved in a variety of important functions in eukaryotic cells. One of the evolutionarily conserved functions of introns is their role in regulating transcription of genes that harbors them. This effect of a splicing-competent intron on transcription is known as ‘Intron-Mediated Enhancement of transcription’ (IME). It has been observed that the intron-containing genes are often transcribed more efficiently than non-intronic genes. However, the molecular mechanism underlying IME in budding yeast and higher eukaryotes is not entirely clear, and that forms the basis of my thesis. To address this issue, I have organized my research project into three specific aims. The primary objective of the first aim was to investigate the mechanism of enhancement of transcription by an intron. I found that the intron-mediated enhancement in budding yeast is dependent on the gene assuming a unique architecture called gene loop. In the second aim, I explored the molecular basis underlying enhancement of transcription by the intron-facilitated gene loop. In the third aim, I determined the effect of position of an intron within a gene on its transcription regulatory potential.
In the first aim, I randomly selected six genes and compared their transcription in the presence and absence of an intron by strand-specific TRO approach. I observed a sharp decline in transcription in the absence of intron. Furthermore, I found that the gene assumed a looped conformation in the presence of an intron. Intron-dependent gene loop was stabilized by three types of interactions; the promoter-terminator, the promoter-5’ splice site and the terminator-3ꞌ splice site interactions. More importantly, I found that the intron-dependent enhancement was completely dependent on gene looping as no enhancement of transcription by an intron was observed in the looping defective mutant.
In the second aim, I investigated how the intron-mediated gene looping regulates transcription. My hypothesis was that intron-mediated gene looping confers directionality, and thereby enhances transcription. During initiation of transcription, the promoter-bound RNAP II has a tendency to transcribe both the downstream coding region in sense direction producing mRNA, as well as the upstream non-coding region in the anti-sense direction producing uaRNA (upstream-antisense RNA). However, there are certain checkpoints in the cell that allows the selective transcription in the sense direction over anti-sense direction, hence maintaining promoter directionality. My results reveal that the intron-dependent gene looping facilitates the recruitment of termination factors in the promoter-proximal region. These termination factors then selectively terminates the uaRNA synthesis, and hence confers directionality.
My last aim was to see the effect of position of an intron within a gene on transcription of the gene. The generally accepted view is that the intron should be present close to the 5ꞌ end of the gene to bring about enhancement of transcription. Whether the presence of intron near the 3ꞌ end of the gene results in enhancement of transcription in yeast was unclear. To address the issue, I inserted the intron in the intron-less version of IMD4 gene at three positions, and showed that even the terminator-proximal intron can enhance transcription. Till now my results have shown that the terminal-proximal intron also enhances transcription in a way similar to the promoter-proximal intron, that is, by conferring promoter directionality.
Agarwal, Neha Agarwal, "An Investigation Into The Molecular Basis Underlying Enhancement Of Transcription By The Intron In Budding Yeast" (2016). Wayne State University Dissertations. 1616.