Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 212-225). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph.D.)--Bowling Green State University, 2008. / Document formatted into pages; contains x, 126 p. : ill. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Alan M. Lambowitz. Vita. Includes bibliographical references.

Thesis (M.S.)--Ohio University, August, 2003. / Title from PDF t.p. Includes bibliographical references (leaves 114-117)

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Biochemistry. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M.S.)--Rochester Institute of Technology, 2007. / Typescript. Includes bibliographical references (leaves 66-67).

Alternative pre-messenger RNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call 'alternative-conserved exons' (ACEs) from other orthologous human/mouse exons, and integrate these features into an exon classification algorithm, ACEScan. Genome-wide analysis of annotated orthologous human-mouse exon pairs identified ~2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of ACEScan predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons which had EST or cDNA evidence of AS in human but were not predicted ACEs, and was never observed for eleven negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve reading frame, and less likely to disrupt protein domains than other AS events, and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing and development. Our results also imply that the vast majority of AS events represented in the human EST databases are not conserved in mouse, and therefore may represent aberrant, disease- or allele-specific, or highly lineage-restricted splicing events.

Rhabdomyosarcoma (RMS) is a highly malignant pediatric cancer that is the most common form of soft tissue tumors in children. RMS cells have many features of skeletal muscle cells, yet do not differentiate. Thus, our studies have focused on the molecular defects present in these cells that block myogenesis. We have found MEF2D is absent in RMS cell lines representing both major subtypes of RMS and primary cells derived from an embryonal RMS mice model. We have shown that the down regulation of MEF2D is a major cause for the failure of RMS cells to differentiate. We find MEF2D cannot bind to muscle specific gene promoters. Exogenous expression of MEF2D activates muscle specific luciferase constructs, upregulates p21 expression and increases muscle specific gene expression including the expression of myosin heavy chain, a marker for skeletal muscle differentiation. Restoring expression of MEF2D also inhibits proliferation, cell motility, anchorage independent growth in vitro, and tumor growth in vivo by xenograft assay. We also have found MEF2C is deregulated in rhabdomyosarcoma with the aberrant alternative splicing. We have shown that exon α in MEF2C is aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 being highly downregulated in RMS cells compared with normal myoblasts. We find that MEF2Cα1 is the ubiquitously expressed isoform which exhibits no myogenic activity and that MEF2Cα2, the muscle specific MEF2C isoform, is required for efficient differentiation. Compared with MEF2Cα2, MEF2Cα1 more strongly interacts with and recruits HDAC5 to myogenic gene promoters to repress muscle specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increases myogenic activity and promotes differentiation in RMS cells. We have also identified a serine protein kinase, SRPK3, which is downregulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage independent growth of RMS cells. The NAC complex performs many diverse biological functions, and the deregulation of its subunits has been correlated with many cancers. We sought to understand the function of the NAC complex in normal myogenesis and tumor progression in rhabdomyosarcoma cells. We found that the muscle specific subunit of the NAC complex, skNAC, which is the alternatively spliced isoform of NACα, was induced in normal cells and downregulated in RMS cells, while BTF3, also known as NACβ, was induced in normal cells and severely downregulated in RMS cells. We also showed that skNAC associated with muscle specific promoters together with BTF3 in differentiated normal cells, and this association was dependent on the expression of BTF3. We further investigated the involvement of skNAC in RMS progression. We found that the muscle specific expressed methyltransferase Smyd1 was nuclear localized in RMS cells and its interaction partner skNAC was switched with corepressors (HDAC1 and TBX2). We also confirmed the expression of skNAC was regulated by the splicing factor kinase SRPK3 and overexpression of SPRK3 induced skNAC expression and muscle differentiation in RMS cells. We also confirmed the overexpression of BTF3 in patient RMS tumors and depletion of BTF3 induced apoptosis in RMS cells and decrease RMS cell survival. BTF3 depletion also sensitized TRAIL induced cell apoptosis in RMS cells. However, BTF3 played a different role in normal cells. Deletion of BTF3 in C2C12 cells does not induce cell apoptosis, which suggests BTF3 functions as an anti-apoptosis factor in RMS cells and could be used as a cancer specific therapeutic target in RMS cells.

The misregulation of post-transcriptional mechanisms has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these misregulated RNA pathways is essential to fully understand the disease mechanisms, identify novel biomarkers, and to develop effective therapies. In this thesis, I present three manuscripts that investigate the mechanisms behind the post-transcriptional misregulation of RNA in human disease, with a focus on pre-mRNA splicing. In the first manuscript (Bondy-Chorney et al., 2016a), we investigated the role of Staufen1 (Stau1) in splicing regulation in the neuromuscular disorder Myotonic Dystrophy Type 1 (DM1). Here we report the first insights into the mechanism that Stau1 uses to regulate the alternative splicing of INSR exon 11 through an interaction with Alu elements located in intron 10. Moreover, using a high-throughput RT-PCR screen, we uncovered a number of additional Stau1-regulated alternative splicing events in both wild-type and DM1 myoblast cell lines. As Stau1 is known to be aberrantly upregulated in DM1 skeletal muscle, our findings suggest that Stau1 acts as a disease modifier in this disorder. The second manuscript (Sanchez, Bondy-Chorney et al., 2015), describes a novel role of the protein methyltransferase Coactivator-Associated Methyltransferase-1 (CARM1), a protein found to be overexpressed in Spinal Muscular Atrophy (SMA). We found that CARM1 can act as a mediator in the nonsense-mediated decay pathway (NMD) and associated UPF1 to promoted its occupancy on PTC-containing transcripts. We identified a subset of natural non-PTC containing NMD targets that were dependent on CARM1, a number of which were misregulated in SMA. This work uncovered a novel role for CARM1 in the NMD pathway and revealed that defective targeting of PTC-containing mRNAs should be included in the complex array of molecular defects associated with SMA. Finally, the third manuscript (Bondy-Chorney et al., – in prep) examines the alternative iv splicing regulation of the Protein Arginine Methyltransferase PRMT1 exon 2, an event shown to alter the growth, survival, and invasion of breast cancer cells. Here, we used an RNA interference (RNAi) RT-PCR screen to uncover several splicing proteins that regulate the inclusion of exon 2, several of which we found to be misregulated in a panel of breast cancer cell lines and patient tumours. These findings confirmed that the inclusion of PRMT1 exon 2 was regulated by alternative splicing via splicing factors that are altered in breast cancer. Moreover, depletion of one of these splicing factors, RALY, resulted in a decrease in the motility and invasive potential of an aggressive breast cancer cell line. These three manuscripts represent a collection of work focused on elucidating the mechanisms involved in post-transcriptional misregulation of RNA in three diverse human diseases. Taken together, the data presented here highlight the broad impact that proteins, such as Stau1 and CARM1, can have in neuromuscular disorders. Moreover, we also uncovered novel misregulation of splicing proteins that alter alternative splicing patterns in breast cancer. Elucidating these mechanisms is of the highest importance in order to identify potential new and effective treatment avenues.

Abstract Type IV collagen is only found in basement membranes, where it is the major structural component, providing a framework for the binding of other basement membrane components and a substratum for cells. The type IV collagen molecule is triple-helical and composed of three a chains which exist as six distinct forms (α1 - α6). Abnormalities in this basement membrane collagen structure and function are connected to both inherited and acquired diseases. Alport syndrome is a hereditary kidney disease associated with extrarenal complications, such as sensorineural deafness and eye abnormalities. The disease is caused by mutations in the COL4A3, COL4A4 and COL4A5 genes, coding for the type IV collagen α3, α4 and α5 chain genes, respectively. About 85% of the Alport syndrome cases are X-linked dominant, caused by mutations in the COL4A5 gene. In order to develop a basis for automated mutation analysis of the COL4A5 gene, previously unknown intron sequences flanking exons 2 and 37 were determined. Intron sequences flanking the other 49 exons were expanded from 35 to 190, and additionally, two novel 9 bp exons (exons 41A and 41B) were characterized in the large intron 41. In addition to optimization of the PCR amplification and sequencing conditions for all 51 exons and exon flanking sequences, optimization for the 820 bp promoter region and for the two novel exons was performed as well. Mutations were found in 79 unrelated patients of the 107 studied. This gives a high mutation detection rate of almost 75% in comparison with 50%, at its best, in other extensive mutation analyses of the COL4A5 gene using SSCP analysis. None of the mutations involved the promoter region or exons 41A and 41B. Circulating antibodies against basement membrane components have been recognized in some autoimmune diseases. Goodpasture syndrome is a rare autoimmune disease characterized by progressive glomerulonephritis and pulmonary hemorrhage. The target of the antibodies in this disease has been shown to be the noncollagenous NC1 domain of type IV collagen α3 chain. For unknown reasons, a minority of Alport syndrome patients also develops antibodies against α3 and α5 chains after renal transplantation with manifestation of severe anti-GBM disease. In order to investigate the antibodies both in Goodpasture and Alport syndrome, the NC1 domains of all six type IV collagen chains were produced as recombinant proteins in bacterial and mammalian expression systems, and an ELISA method was developed for antibody detection. Antibodies were found in both syndromes, interestingly also in Alport syndrome patients without the anti-GBM disease. The results of this work have a significant clinical value by providing for the first time complete, effective DNA-based analysis of all exon/intron and promoter regions of the COL4A5 gene in Alport syndrome.