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Purification, Identification and Characterization of Mammalian Endoribonucleases that Degrade C-myc MRNA in Vitro

出版	University of Northern British Columbia, 2007
URL	http://books.google.com.hk/books?id=GeJczgEACAAJ&hl=&source=gbs_api

註釋There is increasing evidence that mammalian endoribonucleases play a significant role in the degradation of messenger RNA (mRNA) and are key players in the regulation of gene expression particularly under conditions of cellular stress. One of the major challenges, however, is identifying these enzymes and assessing their significance within the context of normal/basal levels of gene expression. To this end, our understanding of this diverse set of enzymes, the mechanisms by which they operate, and their target substrates, remains somewhat mysterious. Previous studies in this laboratory (Bergstrom et al. 2006) have uncovered a mammalian hepatic-derived endoribonuclease with the ability to degrade c-myc CRD RNA in vitro. However, the identity of the enzyme(s) and the remaining co-purified proteins was not determined. The c-myc transcript is a regulator of cell proliferation, differentiation and apoptosis. Deregulated expression of c-myc prevents differentiation of many cell types, induces apoptosis, induces genomic instability, and is associated with several tumor phenotypes. In addition, there is widespread evidence that mRNA stability plays a critical role in the regulation of c-myc gene expression. The main goal of this thesis was to re-purify and conclusively identify the mammalian hepatic-derived endoribonuclease(s) and the proteins that co-purified with endonucleolytic activity against c-myc CRD RNA in vitro. The first aim of this investigation was to purify and identify enzyme(s) responsible for endoribonucleolytic activity. This portion of the study demonstrates that distinct mammalian proteins with molecular weights corresponding to 17 kDa and 35 kDa, respectively, exhibit endoribonuclease activity against c-myc CRD RNA. The second aim of this study was to further characterize the endoribonuclease(s) and to confirm the identity of the enzyme(s) by immunodepleting native endoribonuclease activity. This study revealed that the 17 kDa endoribonuclease activity was contributed by rat pancreatic ribonuclease A (RNase 1) whereas the 35 kDa endoribonuclease activity was contributed by Apurinic/Apyrimidinic Endonuclease (APE1). The third aim of this study was to test the recombinant 35 kDa endoribonuclease (APE1) for endoribonuclease activity. This study demonstrated that recombinant APE1 does possess endoribonuclease activity and cleaves specifically at dinucleotide UA 1751 of c-myc CRD RNA. This is the first study to demonstrate that APE1 cleaves at a specific site in single-stranded RNA, thus providing a platform for future studies aimed at determining whether APE1 plays a role in the degradation of mRNA within mammalian cells.