The regulation of RNA polymerase III-mediated transcription by p53, AP-1 and JNKs

[摘要] Cellular growth is the accumulation of mass by a cell. This is required in order to ensure the maintenance of cell size upon cell division. As the majority of the dry mass of a cell is protein, protein synthesis is essential for cellular growth. RNA Polymerase III (Pol III) is crucial for protein synthesis as it transcribes the genes encoding 5S ribosomal RNA and the tRNAs – key components of the translation machinery. As a consequence, Pol III plays an important role on cell growth and proliferation. Pol III-mediated transcription is highly regulated. Cellular stress usually results in decreased Pol III-mediated transcription, while increased transcription occurs following exposure of cells to mitogens. Understanding how Pol III is regulated is particularly important as loss of regulation is associated with cancer, where Pol III-associated transcription factors and Pol III products are frequently found highly expressed. In this thesis, the role of p53, AP-1 and c-Jun N-terminal kinases (JNKs) in the regulation of Pol III-mediated transcription in humans is examined.p53 is induced in response to stress and modulates the expression of a vast array of target genes. p53 represses Pol III-mediated transcription by binding to the Pol III-specific transcription factor TFIIIB and inhibiting polymerase recruitment to Pol III-target genes. In Chapter Three, it is demonstrated that repression of Pol III-mediated transcription is not the universal response to p53 upregulation. Treatment with the chemotherapeutic drug doxorubicin induces p53 robustly, and results in rapid reduction in tRNA levels but this is not dependent upon the presence of p53. Indeed, it is demonstrated that stress does not always result in the repression of Pol III-mediated transcription. Exposure of cells to ultraviolet light leads to an increase in tRNA levels. This demonstrates the complexity of the regulation of Pol III-mediated transcription in response to stress.Together, c-Jun and c-Fos produce one form of the transcription factor AP-1. AP-1 mediates cell proliferation and cell death in response to mitogenic stimuli and stress via the binding and activation of target genes. c-Jun and c-Fos have been found binding Pol III target genes. In Chapter Four, their occupancy at tRNA genes is examined in detail and found to be associated with active genes. While AP-1 binding motifs are found at a subset of tRNA genes, this is not found to be associated with c-Jun or c-Fos occupancy. Instead, recruitment to tRNA genes may occur via the association with Pol III-specific transcription factors that is observed.JNKs can regulate Pol III-mediated transcription by altering the level of the Pol III-specific transcription factor TFIIIB. JNKs also phosphorylate and activate c-Jun and other transcription factors, such as STAT1 and ATF2, found at Pol III target genes. The role of these transcription factors at tRNA genes has not been determined. However, in Chapter Five, it is demonstrated that JNKs may regulate Pol III-mediated transcription independently of regulating TFIIIB levels. Treatment of U2OS cells with a JNK inhibitor results in rapid reduction of tRNA levels resulting from reduced expression of tRNA genes. However, the levels of TFIIIB subunits TBP and Brf1 are unaffected. It is examined whether this effect may occur through a JNK target at tRNA genes or via another possible route, such as the phosphorylation of the Pol III machinery by JNK. Data in Chapters Four and Five suggest c-Jun and JNK may positively regulate Pol III-mediated transcription. This is consistent with their activation by mitogens, as more Pol III products are required for increased cell growth and proliferation.In conclusion, the data highlights the many pathways that converge to regulate Pol III-mediated transcription, and the complexity that arises as a consequence of this.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] RNA Polymerase III-mediated transcription, p53, tRNA, AP-1, JNK, cell growth.