Date of Award
Doctor of Philosophy (PhD)
Ribosomes are vital to the survival of a cell, as they are directly responsible for the synthesis of proteins, which perform critical cellular functions. As such, majority of the energy reserves in a proliferating cell are expended towards synthesis of ribosomes. Cancer cells, with their enhanced proliferation rates, tend to upregulate ribosome biogenesis in order to meet the demand for increased protein synthesis necessary to sustain rapid proliferation. Many of the oncogenes and tumor suppressors known to be deregulated in cancers are capable of positively and negatively regulating ribosome biogenesis, respectively. The ARF tumor suppressor strongly suppresses ribosome biogenesis, particularly in presence of oncogenic signaling. Furthermore, ARF is capable of negatively regulating multiple oncogenes capable of driving tumorigenesis partly through the ribosome biogenesis pathway. As ARF loss is a frequent occurrence in cancer cells, delineating the ARF-regulatory network and determining the impact of ARF loss on this network can give significant insight into the biology of ARF-deficient tumor cells. Expression of the RNase III enzyme, Drosha, has been reported to have prognostic value in multiple cancers. However, Drosha expression appears to have a dual nature in tumorigenesis, as both overexpression and loss of Drosha have been reported to have tumorigenic functions. Although the mechanistic basis of this apparent duality are not yet known, gaining a deeper understanding of Drosha's functional capabilities can give us an insight into its role in tumorigenesis. Drosha performs critical functions in biogenesis of multiple RNA species within the cell, including ribosomal RNA (rRNA), micro RNA (miRNA) and messenger RNA (mRNA). Drosha's role in miRNA biogenesis is the most studied and characterized aspect of its functions and can explain the tumor suppressive aspect of its dual nature; a global decrease in miRNAs has been reported to be part of tumor progression, and loss of Drosha has the potential to significantly deplete mature miRNA population within the cell. However, how overexpression of Drosha can drive tumorigenesis remains to be studied. As enhanced ribosome biogenesis is another feature of cancer cells and Drosha has been shown to aid in processing of r RNA, Drosha's role in ribosome biogenesis pathway has the potential to function in an oncogenic manner. Therefore, further characterization of Drosha's role in ribosome biogenesis can significantly enhance our understanding of its contribution to tumorigenesis. Recent studies in mouse cell lines revealed that ARF tumor suppressor is capable of negatively regulating Drosha expression in a translation-dependent manner. Given the entrenched role of ARF in inhibiting ribosome biogenesis, I hypothesized that ARF's ability to regulate Drosha could impact Drosha's functions in ribosome biogenesis pathway. I further hypothesized that Drosha overexpression could function in a pro-proliferative manner through the ribosome biogenesis pathway. The data presented in this Dissertation reveals that human p14ARF is capable of regulating Drosha protein expression in a dynamic and localized fashion; loss of ARF increases over all cellular Drosha protein levels and also the localization of Drosha to the nucleolus. ARF potentially regulates nucleolar localization of Drosha by sequestering it away from nucleolus, as we found that ARF immunoprecipitated with Drosha in RNA-independent manner. Furthermore, loss of ARF enhances ribosome biogenesis both at the level of 47s rRNA transcription and processing. Association of Drosha with precursor rRNAs was also enhanced in absence of ARF, suggesting that enhanced nucleolar localization of Drosha upon ARF loss contributes to rRNA processing. Drosha overexpression by itself was able to increase ribosome biogenesis, with a modest increase in 47s rRNA transcription and a faster accumulation of 28s and 18s rRNAs. Drosha overexpression led to an increase in ARF expression, although this induction of ARF was not sufficient to inhibit Drosha's ability to enhance ribosome biogenesis and cell proliferation. However, overexpression of ARF negated proliferative enhancement induced by Drosha overexpression. These results point towards a cross-regulatory loop between ARF and Drosha, with functional impact on ribosome biogenesis.
Chair and Committee
Jason D. Weber
Zhongsheng You, Sergej Djuranovic, Audrey McAlinden, Susana Gonzalo
Yaddanapudi, Sree Chandana Sridhar, "The Human ARF Tumor Suppressor Regulates Drosha Nucleolar Localization and rRNA Processing Activity" (2016). Arts & Sciences Electronic Theses and Dissertations. 1016.