Pre-mRNA molecules in humans contain mostly short internal exons flanked by long introns. To explain the removal of such introns, recognition of the exons instead of recognition of the introns has been proposed. This thesis studies this exon definition mechanism using a bottom-up approach. To reduce the complexity of the system under study, this exon definition mechanism was addressed using designer exons made up of prototype sequence modules of our own design (including an exonic splicing enhancer or silencer). Studies were performed in vitro with a set of DEs obtained from random combinations of the exonic splicing enhancer and the exonic splicing silencer modules. The results showed considerable variability both in terms of the composition and size of the DEs and in terms of their inclusion level. To understand how different DEs generated different inclusion levels, the problem was divided into understanding separately parameters varied between DEs. Subsequent studies focused on each of three parameters: size, ESE composition and ESS composition. The final objective was to be able to combine their effects to predict the inclusion levels obtained for the "random" DEs mentioned previously. To complement this experimental approach an equation was generated in two stages. First a general "framework" equation was obtained modeling a necessary exon definition complex that enabled commitment to inclusion. This equation used rates for the formation and dissociation of this complex without elaborating on the details for how those rates came about. In the second stage, however, formation and dissociation were modeled using novel but intuitive ideas and these models were combined into a final equation. This equation using the single-parameter perturbation data obtained previously performed well in predicting the inclusion levels of the "random" DEs. Additionally, both the final equation and the mechanisms proposed align well with results published by other groups. In order to make these results more accessible and to open more opportunities to extend them, an initial attempt is presented to identify the proteins involved in the functionality observed for each of the sequences used.

The human androgen receptor (hAR) is a ligand-activated, nuclear transcription factor. Mutations affecting the formation and/or action of the hAR cause androgen insensitivity syndrome (AIS). The majority of mutations identified to date are within the DNA- and hormone-binding domains; very few have been identified in the transactivational modulatory domain, encoded by exon 1. This work presents an analysis of exon 1 and the 5$ sp prime$-flanking region of the hAR in a set of subjects whose AIS was believed to be caused by a mutation within these regions. Six of twelve strains had a nonsense or frameshift mutation in exon 1; a seventh strain had two missense and one silent substitution; no mutations were identified in the remaining subjects. The two missense mutations were recreated, individually and together, in an hAR complementary DNA (cDNA) expression vector and expressed in heterologous COS-1 cells. Their pathogenicity could not be proven with the system and assays used. In addition, mRNA and protein levels were analyzed and correlated with the identified mutations and the subjects' phenotype.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

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Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xi, 183 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

A new disease of sugar beet, named Fusarium yellowing decline, was recently found in in the Red River Valley of MN and ND. This disease is caused by a novel pathogen named Fusarium secorum. Pathogens such as F. secorum secrete proteins during infection called ‘effectors’ that help establish disease. Since pathogenicity and disease development may depend on effector proteins produced by F. secorum during infection, effector protein identification furthers our understanding of the biology of this important pathogen. A list of 11 candidate effectors was generated previously. In this study, to characterize putative effectors, we developed a transformation system using polyethylene glycol–mediated transformation. Several mutant lines were created with an effector deleted from the genome using a split-marker knock-out strategy. To explore their role in pathogenicity, mutant strains have been inoculated to sugarbeet and compared to WT F. secorum.

Intercellular communication is crucial during animal development and tissue maintenance to ensure that correct patterns of cell types are generated to meet the needs of the organism. During lateral specification, intercellular communication resolves cell fate decisions between equipotent cells, creating fate patterns that are biased by external factors in some contexts, but appear stochastic in others. The Notch signaling pathway mediates lateral specification; small differences in Notch activity are amplified by regulatory feedback loops to robustly differentiate cell fates based on relative levels of Notch activity. It is often unclear how noise in the environment is processed by cells to generate differences in Notch activity that can be translated into stochastic, but robust, cell fate outcomes. The nematode Caenorhabditis elegans contains a simple, Notch-mediated, stochastic lateral specification event; a small, random difference in Notch activity between two cells, the α cells, is amplified so that one α cell assumes Anchor Cell (AC) fate and the other assumes Ventral Uterine precursor cell (VU) fate. Two upstream factors bias the outcome of the AC/VU decision depending on the length of the time interval between the births of the α cells: the relative birth order of the α cells and the onset of expression of the transcription factor HLH-2. It is unknown how these factors create a difference in the relative Notch activity level between the two α cells, and limitations of existing Notch reporters have prevented the direct observation of Notch activity levels required for determining the relationships. In this thesis, I describe a genetically-encoded Sensor Able to detect Lateral Signaling Activity, or SALSA, which uses changes in nuclear Red:Green fluorescence to indicate Notch activity. I demonstrated that SALSA captures expected Notch activity patterns in four paradigms in C. elegans, encompassing both Notch homologs, and reports low levels of Notch activity that were predicted but undetectable with other Notch activity reporters. Using SALSA, I showed that the first-born α cell is able to develop an advantage in Notch activity prior to the birth of the other α cell when the time interval between α cell births is long, but the α cell that gains the Notch activity advantage is random with respect to birth order when the time interval between α cell births is short. These results agree with the current model of the AC/VU decision. I also describe Flexon, a method for the conditional activation of strong gene expression in specific cell lineages using a lox-stop-lox cassette encoded into an artificial exon flanked by two artificial introns. A flexon can be placed into the coding region of a gene to prevent translation of a functional gene product; gene expression is restored to specific lineages through expression of a tissue-specific Cre driver that excises the flexon. I show that flexon can be used to make bright, long-lasting, tissue-specific fluorescent lineage markers. I also showed that the flexon could be used for conditional activation of an endogenous gene by inserting a flexon into rde-1 to severely reduce RNAi activity and restore gene function in specific tissues using Cre drivers.

Over 90% of human genes produce precursor mRNA (pre-mRNA) that undergoes splicing, an RNA processing mechanism. Alternative splicing (AS) of pre-mRNA allows a gene to generate multiple coding and non-coding isoforms by removing introns and ligating distinct exonic combinations. It is a mechanism that plays a major role in driving molecular diversity in mammals. This process is tightly regulated to determine the types and levels of protein products expressed in specific cellular contexts. Cis-acting splicing regulatory elements (SREs) found within the pre-mRNA are recognized and bound by RNA-binding proteins that either assist or interfere with the recruitment of the spliceosome. In the field of splicing, a long-standing goal has been to develop a “splicing code”, or a set of rules to understand the splicing patterns of a gene in a predictable manner. It is essential to highlight the significance of sequence context for SREs and the potential impact that distal intronic elements can have on splice site selection to better understand splicing. Given the importance of sequence context and the involvement of distal intronic regions in splicing, future approaches aimed at identifying SREs should consider these factors.This thesis will describe the process of the identification and validation of two novel distal intronic SREs located in critical disease exons. Importantly, these findings were made by combining experimental and computational approaches and through the development of a high-throughput SRE screening methodology. Chapter 1 will provide a general context for splicing, in particular AS, as an important mechanism among a plethora of RNA regulatory functions. The significance of AS regulation will be explored, as it plays a key role in the occurrence of physiological events and incorrect regulation can trigger disease. I will also introduce several methods used to study SREs, with experimental efforts primarily focusing on exonic and proximal intronic sequences. Additionally, as mis-splicing is associated with disease, there is a high interest in modulating splicing with novel therapeutic interventions, the development of which benefits from an increased understanding of SREs. Specifically, I will provide a landscape view of the splicing research field for the genes spinal muscular atrophy 2 (SMN2) and microtubule-binding protein tau (MAPT), given their relevance to our discoveries. As there is currently a paucity of high-throughput methods for studying SREs, especially those that allow for analysis of SREs in a near-native sequence context, I will introduce the CRISPR-Cas system (dCas13d) as a potential splicing modulator. This system will form the foundation for developing a tool to help us understand splicing regulation. Chapter 2 will discuss the discovery of a distal SRE regulating MAPT exon 10 splicing divergence in the primate lineage. Our lineage-specific AS analysis found that MAPT exon 10 shows a two-step evolutionary shift in the Catarrhine and hominoid lineages. The previously identified splicing regulatory elements cannot explain this evolutionary shift. Instead, a key splicing factor, muscleblind-like (MBNL), was found to be a major contributor to the observed splicing pattern divergence. Further mechanistic dissection revealed divergent, distal regulatory sequences in intron 10 that are recognized by MBNL. Based on this finding, we also demonstrated the potential of developing a therapeutically compatible strategy to target the MBNL binding sites by a steric hindrance to modulate exon 10 splicing effectively. Chapter 3 will discuss the development of a method allowing for a more unbiased, high-throughput screening of SREs, including those in the distal intronic regions. This method relies on the nuclease-inactive dRfxCas13d to modulate splicing. We use a dual-color fluorescent splicing reporter to identify the impact of splicing in a high-throughput manner. For our proof of concept, we use SMN2 exon 7 to identify SREs that influence the splicing of this exon and corroborate our findings with known SMN2 SREs. We performed a screen on the SMN2 dual-color splicing reporter using a gRNA library and obtained highly reproducible results. The screen also correctly identified gRNAs targeting known SREs, including the well-studied exonic regions and the downstream ISS-N1 element, the target of the ASO therapeutic known as nusinersen. Importantly, this screen also discovered novel splicing inhibiting gRNAs in a more distal region of the downstream intron, suggesting that a robust splicing enhancer was targeted. Previous studies likely overlooked this region due to its distance from the exon. This novel approach allows for the simultaneous screening of sizeable genetic regions using a large-scale gRNA library.

Estudamos 32 crianças com HC devido à agenesia ou ectopia tireoideana para mutações no PAX8 e 30 crianças com hipoplasia da tireóide para mutações no rTSH. Todos os exons de ambos os genes foram amplificados a partir do DNA genômico, seguido por seqüenciamento direto. Encontramos, em dois pacientes com ectopia, duas alterações no gene PAX8, uma no promotor, e outra no exon um. Os outros indivíduos estudados apresentaram as seqüências codificáveis dos genes PAX8 e rTSH normais. Em relação ao caráter funcional e ensaios de luciferase verificamos que no promotor a resposta transcricional diminuiu significativamente na presença de TSH, por um mecanismo dependente de cAMP / We studied 32 children with hypothyrodism (CH) from thyroid agenesis or ectopia for PAX8 mutations, and 30 children with thyroid hypoplasia for rTSH mutations. All exons of both genes were amplified from the genomic DNA, then sequenced directly. We found two alterations in the PAX8 gene in two patients, one in the promoter and the other in exon one. The other children had normal sequences in both PAX8 and rTSH genes. In relation to functional character and luciferase assays, we verified that transcriptional response was significantly reduced in the presence of TSH by a cAMP dependant mechanism

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 111-119). Also available on the Internet.