All ETDs from UAB

Advisory Committee Chair

Keith E Giles

Advisory Committee Members

David A Schneider

Christopher Klug

Michael R Crowley

Xinyang Zhao

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The ribosome has the major role of synthesizing almost all the proteins of the cell. One of the main structural components of the ribosome are the ribosomal RNAs 18S, 28S, 5.8S and the 5S which are bound by numerous proteins to make up a fully functional unit. In the human genome there are approximately 400 copies of the rRNA gene, which contains all but the 5S rRNA in one long transcription unit. This gene is found in tandem repeats on the acrocentric arm of chromosomes 13, 14, 15, 21 and 22. It is critical that this gene is tightly regulated to keep up with the demands of cellular growth and maintenance. Many studies have shown a correlation between rRNA biogenesis and tumor progression of cancer cells pointing out the importance of regulating rRNA transcription. Many factors aid in the regulation of the rRNA gene. One of the most important proteins that aids in transcriptional regulation of the rRNA gene is upstream binding transcription factor (UBTF). It regulates transcription by binding to the promoter and recruiting RNA polymerase I (Pol I), the polymerase responsible for synthesis of the 18S, 5.8S and 28S rRNAs. Selectivity factor 1 (SL-1) is a protein complex which stabilizes UBTF at the promoter and together they initiate Pol I transcription. The loss of UBTF at the promoter correlates very closely with the down regulation of rRNA synthesis. Here we show that when stem cells are exposed to differentiation factors, the localization of UBTF at the promoter of the rRNA gene decreases. This decrease is mirrored by a drop in synthesis rate of rRNA. We hypothesized that down regulation of rRNA is an essential step for cell differentiation. To explore this idea we treated stem cells with CX-5461, which is a Pol I specific inhibitor, and observe that down regulation of rRNA synthesis alone leads to differentiation. These results indicate that the regulation of rRNA synthesis is important in the differentiation ability of cells. We also demonstrate that UBTF is able to bind to other genes outside the nucleolus and that loss of UBTF at these genes correlates with upregulation of these genes during differentiation. This data suggests that UBTF may play a role in regulating other genes outside of the rRNA gene during differentiation. We also propose a new mechanism in which rRNA maybe cleaved co-transcriptionally as a means of regulating total rRNA levels. This mechanism involves Ago2 targeting rRNA through a small RNA independent interaction. Ago2 not only binds to snoRNAs but it also localizes to rRNA, which correlates to snoRNA modification sites as well as cleavage site. Knock down of Ago2 leads to an increase in total nascent rRNA and a reduction in cleavage of rRNA. This data suggests that Ago2 is localizing to the rRNA through a snoRNA mediated mechanism and regulating rRNA levels co-transcriptionally.

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