The Integrator complex (INT) is a metazoan-specific group of proteins associated with RNA polymerase II (Pol II) that has important functions in the 3'-end processing of noncodng RNAs, including uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA). Recently, INT has also been reported to be involved in Pol II transcriptional regulation of protein-encoding genes. INT contains at least 14 subunits, but the function of each subunit is difficult to predicted, because most subunits lack identifiable domains and display little similarity with other proteins. The endonuclease activity of INT is carried out by its subunit 11 (IntS11), which belongs to the metallo--lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with INT subunit 9 (IntS9) through their C-terminal domains (CTDs). This dissertation describes the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and summaries the structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded -sheet with four strands from IntS9 CTD and five from IntS11 CTD. Highly conserved residues are located in the interface between the two CTDs. The structural observations on the complex are confirmed by yeast two-hybrid assays and coimmunoprecipitation experiments. Functional studies demonstrate that the Int9-IntS11 interaction is crucial for proper INT function in snRNA 3'-end processing. The dissertation also presents the structural studies of a newly found mammalian mRNA deNADding enzyme, Nudt12. We determined the crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6-Å resolution. The structure provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Previous studies have reported that NAD-capped mRNAs in mammalian cells are hydrolyzed by the DXO deNADding enzyme. Together with biochemical and functional studies, we demonstrate that Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targets different RNAs.

Chronic Myeloid Leukaemia (CML) is a clonal haemopoietic stem cell (HSC) disorder characterised by the presence of a disease-specific gene, BCR-ABL, which leads to the production of a bcr-abl mRNA transcript. CML is an ideal candidate for gene therapy using ribozymes (Rz), catalytic RNA molecules that cleave and inactivate target RNA in a sequence specific manner. Limited data is available on the activity of ribozymes in human CML cells. In this study, hammerhead ribozyme sequences directed against the b3a2 bcr-abl mRNA sequence (Rz6-Rz10) were cloned into several retroviral vectors. Initial experiments using MSCVHSA based retroviral constructs failed to express the sequences in cell lines. Rz cDNA fragments were then cloned into an LNL6 based retroviral vector (LGL1) encoding a GFP reporter gene and stable LGLRz constructs produced. Using cell sorting, high-titre PA317 producer cell line clones were isolated. Transcriptional silencing of the LGLRz6 producer cell line occurred with prolonged culture, with partial reversal on treatment with the demethylating agent 5' azacytidine. To assess the activity of these constructs in human cells, CD34+ HSC were isolated from newly diagnosed b3a2 Ph+ CML patients. Cells were transduced with either control LGL vector or the LGLRz6 construct. Transduced human cells were sorted based on GFP expression and placed into long-term HSC culture (LTC-IC assays). Using a common cDNA, RT-PCR was performed to detect the expression of both the transgene and bcr-abl in individual colonies derived from the LTCIC assay at various time points, allowing assessment of the effect of transgene expression on bcr-abl expression. LGLRz transgene expression was detectable for up to 6 weeks in culture. Colony RT-PCR results from 3 patients showed that expression of the LGLRz6 construct was associated with decreased bcr-abl expression. It also appeared that the reduced bcr-abl expression decreased the proliferation of Ph+ cells leading to their loss from culture. In summary, these results appear to show an effect of a retroviral vector containing a bcr-abl Rz sequence on human CML HSC. Targeting of bcr-abl remains a valid therapeutic goal in the Imatinib era, particularly if problems related to effective ribozyme delivery and targeting can be overcome.

Chronic Myeloid Leukaemia (CML) is a clonal haemopoietic stem cell (HSC) disorder characterised by the presence of a disease-specific gene, BCR-ABL, which leads to the production of a bcr-abl mRNA transcript. CML is an ideal candidate for gene therapy using ribozymes (Rz), catalytic RNA molecules that cleave and inactivate target RNA in a sequence specific manner. Limited data is available on the activity of ribozymes in human CML cells. In this study, hammerhead ribozyme sequences directed against the b3a2 bcr-abl mRNA sequence (Rz6-Rz10) were cloned into several retroviral vectors. Initial experiments using MSCVHSA based retroviral constructs failed to express the sequences in cell lines. Rz cDNA fragments were then cloned into an LNL6 based retroviral vector (LGL1) encoding a GFP reporter gene and stable LGLRz constructs produced. Using cell sorting, high-titre PA317 producer cell line clones were isolated. Transcriptional silencing of the LGLRz6 producer cell line occurred with prolonged culture, with partial reversal on treatment with the demethylating agent 5' azacytidine. To assess the activity of these constructs in human cells, CD34+ HSC were isolated from newly diagnosed b3a2 Ph+ CML patients. Cells were transduced with either control LGL vector or the LGLRz6 construct. Transduced human cells were sorted based on GFP expression and placed into long-term HSC culture (LTC-IC assays). Using a common cDNA, RT-PCR was performed to detect the expression of both the transgene and bcr-abl in individual colonies derived from the LTCIC assay at various time points, allowing assessment of the effect of transgene expression on bcr-abl expression. LGLRz transgene expression was detectable for up to 6 weeks in culture. Colony RT-PCR results from 3 patients showed that expression of the LGLRz6 construct was associated with decreased bcr-abl expression. It also appeared that the reduced bcr-abl expression decreased the proliferation of Ph+ cells leading to their loss from culture. In summary, these results appear to show an effect of a retroviral vector containing a bcr-abl Rz sequence on human CML HSC. Targeting of bcr-abl remains a valid therapeutic goal in the Imatinib era, particularly if problems related to effective ribozyme delivery and targeting can be overcome.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 106-118).

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 129-140)

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, December 2000. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Department of Chemistry, March 2003. / Includes bibliographical references. Also available on the Internet.

DExD/H-box proteins are a diverse class of proteins that are implicated in RNA and RNP remodeling. They have sequence homology to DNA helicases and share conserved ATPase domains, suggesting that they use the energy of ATP binding and hydrolysis to mediate conformational rearrangements in RNAs. In the past, the action of DExD/H-box proteins has been characterized primarily on simple model substrates such as small RNA duplexes. It is not known how DExD/H-box proteins manipulate structured RNA, what determines target specificity and what molecular events follow their action. Here, using the well-characterized Tetrahymena group I intron ribozyme, I performed kinetic and thermodynamic studies to understand the mechanism of CYT-19 and related DExD/Hbox proteins. CYT-19 has been shown previously to facilitate the folding of several group I and group II introns. I demonstrated that CYT-19 acts as a chaperone, accelerating the re-folding of a long-lived misfolded species of the Tetrahymena group I ribozyme to its native state. Further characterization of this reaction gave insights into how CYT-19 achieves this action; CYT-19 partially unfolds the misfolded ribozyme and allows it to fold again along the same pathway that exists in the absence of CYT-19. In addition to acting on the misfolded state, CYT-19 also acts on the native state, but this action is largely obscured under stabilizing conditions for the native state because the action is inefficient under such conditions. However, under conditions where the native state is destabilized, the native ribozyme was indeed shown to be partially unfolded by CYT-19. By acting on either species, CYT-19 sets up a steady state of unfolding, and the distribution is shifted from equilibrium to kinetic control, increasing the relative populations of conformations that are kinetically preferred during folding. The efficiency of action seems to correlate with the stability of the ribozyme. These activities are not restricted to CYT-19; the DExD/H-box proteins Mss116p and Ded1 were demonstrated to possess similar activities. Together, these studies give important insights into the mechanisms of action for this ubiquitous class of proteins and have implications for all structured RNAs in cells. / text

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.