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Regulation of U1 snRNP / 5' splice site interactions during pre-MRNA splicing in Saccharomyces cerevisiaeStands, Leah Rae, January 2003 (has links)
Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xiv, 236 p.; also includes graphics (some col.) Includes bibliographical references (p. 215-236). Available online via OhioLINK's ETD Center
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An investigation of splicing-dependent transcriptional checkpointsThelakkad Chathoth, Keerthi January 2013 (has links)
Pre-mRNA splicing and other RNA processing events occur co-transcriptionally. High resolution kinetic studies performed in our lab showed splicing-dependent RNA Pol II (RNA polymerase II) pausing near the 3’ splice site of a reporter gene. Pausing requires splicing, as mutations that block splicing lead to loss of pausing, and restoring splicing restores pausing. It was proposed that RNA Pol II pausing may occur at splicing-dependent transcriptional checkpoints. In this study, I aimed to search for splicing helicases that might couple splicing with transcription. The ts alleles prp5-1 and prp16-2 were found to cause transcription defects. These genes encode RNA helicases that were reported to act as fidelity factors during splicing. In vivo RNA labelling and RT-qPCR experiments performed with these temperature-sensitive mutants demonstrated reduced transcription coinciding with the splicing defect at restrictive temperature. Furthermore, RNA Pol II ChIP analysis showed polymerase accumulating over intron-containing genes in both mutants. ChIP analysis using antibodies specific to the phosphorylation status of the CTD (Carboxy Terminal Domain) of RNA Pol II, revealed that the apparently stalled polymerase is hyper-phosphorylated at serine 5. Intriguingly, prp8-R1753K, a ts allele of PRP8, a non-helicase splicing factor mutant also showed reduced nascent RNA synthesis but no RNA Pol II accumulation. To elucidate the reason for the observed RNA Pol II accumulation and to identify a possible splicing-dependent transcriptional checkpoint factor, prp5-1 was investigated further. RNA Pol II ChIP-Seq analysis verified that maximum enrichment genome-wide occurred on introns at restrictive conditions in prp5-1, supporting the earlier observation. Furthermore, the double mutant strain cus2Δprp5-1 abolished the RNA Pol II accumulation observed in prp5-1 at restrictive temperature and restored transcription. Recreating a stalled spliceosome in a U2 mutant strain also showed RNA Pol II accumulation in the presence of Cus2p, as observed in prp5-1. My observations suggest a link between transcription and monitoring of splicing and indicate that Cus2p, a U2 snRNP associated protein, could be a checkpoint factor in transcription prior to pre-spliceosome formation. I speculate that fidelity factors may impose transcriptional checkpoints at different stages of splicing.
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Targeted mutagenesis and functional analysis of CWC25 Splicing Factor in Rice via CRISPR/Cas9Kababji, Ahad M. 11 1900 (has links)
Pre-mRNA splicing is the most critical process in gene expression regulation across eukaryotic species. This reaction is carried out by the spliceosome, a large, dynamic, and well-organized ribonucleoprotein complex. The spliceosome is composed of five major small nuclear RNAs and an excessive number of associated protein factors. Many protein splicing factors bind and release during splicing to assist the assembly and the modulation of many RNA structures and proteins within the spliceosome. CWC25 is a splicing protein factor that functions in modulating the conformational structure of the spliceosome at the first transesterification reaction. CWC25 binds with its N-terminus to the major groove of the catalytic spliceosome triggering the spliceosome activity. Here, we employed CRISPR/Cas9 genome engineering system for targeted mutagenesis to generate CWC25 functional knock-out mutants to understand its molecular function, contribution to splicing regulation and implication in fine-tuning responses to abiotic stress in rice. Our genotyping analysis of the OsCWC25 locus revealed the presence of two mono-allelic and 18 bi-allelic mutant lines. Phenotypic analysis of these mutants, including germination and root inhibition assays, showed that the cwc25 mutants are oversensitive to abiotic stresses such as ABA and salinity. Our data demonstrate that CWC25 plays an important role in regulating plant responses to abiotic stresses.
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Análise de splicing alternativo utilizando dados de sequências expressas / Analysis of alternative splicing by using expressed sequence dataVillagra, Ulises Maximiliano Mancini 02 October 2009 (has links)
O splicing alternativo é um processo pelo qual os exons de um transcrito primário são ligados de diferentes maneiras durante o processamento do RNA, levando à síntese de proteínas distintas. Compreende um importante mecanismo na expressão gênica de eucariotos, responsável pelo aumento da diversidade proteômica e, portanto da capacidade codificante do genoma. Diferentes mecanismos parecem afetar a regulação do splicing alternativo, incluindo estresse metabólico. No presente estudo, foi realizada uma análise detalhada de sequências ORESTES de tecidos de cabeça e pescoço. Essa análise revelou que o ganho de sequências exônicas é mais freqüente que sua perda, e que a regra GT/AG é predominante em sítios de splicing. Nós observamos que o splicing alternativo geralmente não altera a matriz de leitura, mas pode afetar um domínio protéico e remover ou adicionar novos sítios de fosforilação e glicosilação. Elementos reguladores potenciais e elementos repetitivos foram freqüentes nas sequências alternativas e nas suas vizinhas. A expressão de isoformas de splicing potenciais foi investigada em diferentes tecidos, incluindo sob condições de estresse. Foram validados cerca de 50 eventos de splicing novos em células normais e tumorais. Diversas variantes, tais como aquelas dos genes HNRNPK, ACTN1, BAT3, CEP192, MPV17, PDK1, PRKAR1A, RAG1AP1 e TRIP6 mostraram padrões de expressão distintos em diferentes tipos celulares, em amostras normais e tumorais de pacientes com carcinoma de cabeça e pescoço e, em alguns casos, em diferentes estágios do tumor. Também foi validado um transcrito novo do gene RIPK2, responsável por codificar uma quinase de serine/treonine que ativa a via de sinalização NF-kB, e foi observada uma mudança na expressão dessa variante em resposta ao estresse térmico in vitro. Ainda não está claramente definido se o splicing alternativo é causa ou conseqüência do processo neoplásico. Nossos dados adicionam informações novas a esse tópico e fornecem alguns exemplos que evidenciam a importância do processamento do RNA na regulação da expressão gênica, tanto em condições normais como de doença. / Alternative splicing is a process by which the exons of the primary gene transcript are linked in different ways during RNA processing resulting in distinctive proteins. It is an important mechanism in eukaryotic gene expression that enhances proteome diversity and, therefore, the coding capacity of the genome. Different mechanisms seem affect alternative splicing regulation, including metabolic stresses. In the present study, a detailed informatics analysis of ORESTES sequences from head and neck tissues was performed. This in silico analysis revealed that gain of exon sequences is more frequent than exon skipping and GT/AG rule is predominant in splice sites. We observed that alternative splicing usually does not alter the reading frame but may disrupt a protein domain and remove or add new phosphorylation and glycosylation sites. Repetitive and potential regulator elements were frequent in the alternative sequences or in their neighbors. The expression of putative splicing isoforms was investigated in different tissues, including upon stress conditions. We validated approximately 50 new splicing events in normal and tumor cells. Several variants, such as those from HNRNPK, ACTN1, BAT3, CEP192, MPV17, PDK1, PRKAR1A, RAG1AP1 and TRIP6 genes showed distinctive expression pattern in different cell types, in normal and cancer samples from head and neck carcinoma patients and, in some cases, in different tumor stages. We also validated a new transcript of RIPK2 gene, which codes a serine/threonine kinase that activates the NF-kB pathway, and observed a shift in the expression of this variant as a response to temperature stress in vitro. It is currently not clear whether alternative splicing is the cause or the consequence of the neoplastic process. Our data add new information to this topic and provide some examples on the importance of RNA processing in gene expression regulation, both in normal and disease conditions.
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Análise de splicing alternativo utilizando dados de sequências expressas / Analysis of alternative splicing by using expressed sequence dataUlises Maximiliano Mancini Villagra 02 October 2009 (has links)
O splicing alternativo é um processo pelo qual os exons de um transcrito primário são ligados de diferentes maneiras durante o processamento do RNA, levando à síntese de proteínas distintas. Compreende um importante mecanismo na expressão gênica de eucariotos, responsável pelo aumento da diversidade proteômica e, portanto da capacidade codificante do genoma. Diferentes mecanismos parecem afetar a regulação do splicing alternativo, incluindo estresse metabólico. No presente estudo, foi realizada uma análise detalhada de sequências ORESTES de tecidos de cabeça e pescoço. Essa análise revelou que o ganho de sequências exônicas é mais freqüente que sua perda, e que a regra GT/AG é predominante em sítios de splicing. Nós observamos que o splicing alternativo geralmente não altera a matriz de leitura, mas pode afetar um domínio protéico e remover ou adicionar novos sítios de fosforilação e glicosilação. Elementos reguladores potenciais e elementos repetitivos foram freqüentes nas sequências alternativas e nas suas vizinhas. A expressão de isoformas de splicing potenciais foi investigada em diferentes tecidos, incluindo sob condições de estresse. Foram validados cerca de 50 eventos de splicing novos em células normais e tumorais. Diversas variantes, tais como aquelas dos genes HNRNPK, ACTN1, BAT3, CEP192, MPV17, PDK1, PRKAR1A, RAG1AP1 e TRIP6 mostraram padrões de expressão distintos em diferentes tipos celulares, em amostras normais e tumorais de pacientes com carcinoma de cabeça e pescoço e, em alguns casos, em diferentes estágios do tumor. Também foi validado um transcrito novo do gene RIPK2, responsável por codificar uma quinase de serine/treonine que ativa a via de sinalização NF-kB, e foi observada uma mudança na expressão dessa variante em resposta ao estresse térmico in vitro. Ainda não está claramente definido se o splicing alternativo é causa ou conseqüência do processo neoplásico. Nossos dados adicionam informações novas a esse tópico e fornecem alguns exemplos que evidenciam a importância do processamento do RNA na regulação da expressão gênica, tanto em condições normais como de doença. / Alternative splicing is a process by which the exons of the primary gene transcript are linked in different ways during RNA processing resulting in distinctive proteins. It is an important mechanism in eukaryotic gene expression that enhances proteome diversity and, therefore, the coding capacity of the genome. Different mechanisms seem affect alternative splicing regulation, including metabolic stresses. In the present study, a detailed informatics analysis of ORESTES sequences from head and neck tissues was performed. This in silico analysis revealed that gain of exon sequences is more frequent than exon skipping and GT/AG rule is predominant in splice sites. We observed that alternative splicing usually does not alter the reading frame but may disrupt a protein domain and remove or add new phosphorylation and glycosylation sites. Repetitive and potential regulator elements were frequent in the alternative sequences or in their neighbors. The expression of putative splicing isoforms was investigated in different tissues, including upon stress conditions. We validated approximately 50 new splicing events in normal and tumor cells. Several variants, such as those from HNRNPK, ACTN1, BAT3, CEP192, MPV17, PDK1, PRKAR1A, RAG1AP1 and TRIP6 genes showed distinctive expression pattern in different cell types, in normal and cancer samples from head and neck carcinoma patients and, in some cases, in different tumor stages. We also validated a new transcript of RIPK2 gene, which codes a serine/threonine kinase that activates the NF-kB pathway, and observed a shift in the expression of this variant as a response to temperature stress in vitro. It is currently not clear whether alternative splicing is the cause or the consequence of the neoplastic process. Our data add new information to this topic and provide some examples on the importance of RNA processing in gene expression regulation, both in normal and disease conditions.
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Dscam gene expression in invertebrate immunity : alternative splicing in response to diverse pathogensSmith, Paul Hugh January 2012 (has links)
Invertebrates show enhanced immunity and even specific primed immunity in response to repeat infections, analogous to vertebrate adaptive immunity. Little is known of the mechanism for this phenomenon, or which molecules are involved. A candidate gene for the underlying mechanism for a pathogen-specific response in invertebrate immunity is Down syndrome cell adhesion molecule (Dscam). Dscam can produce thousands of different protein isoforms through the mutually exclusive splicing of many exon variants contained within variable regions of the gene. It is an important receptor of the invertebrate nervous system but has been implicated in having a role in immunity. Dscam has been shown to be involved in phagocytosis across members of the Pancrustacea, and it has been reported to respond in a pathogen-specific manner in mosquitoes and crayfish. In this thesis, I have investigated the splicing of Dscam in response to diverse pathogens in different host species. In the Anopheles mosquito, I cloned and sequenced a fragment of Dscam spanning across two of its variable exon regions to enable me to detect mutually exclusively splice variants and their associations in different treatments (Chapter 2). I discovered that the expression diversity of the hypervariable Dscam is higher in parasite-exposed mosquitoes. In Chapter 3, I extended the study to the more experimentally amenable Drosophila fruit fly. A new Illumina-based sequencing assay was developed and implemented to examine more closely Dscam expression in response to diverse pathogens. The new method successfully quantified non-random expression of Dscam variable exons 4 and 6. I also describe a small but detectable effect of pathogen-exposure on the expression of Dscam exon 4 variants. In Chapter 4, I expanded the work of Chapter 3 to study tissue-specific Dscam expression in response to well-characterised immune elicitors of Drosophila. I describe how exon 4 variants were expressed in a tissue-specific manner, but not exon 6 variants. I also found a small exon 4-by-tissue-by-pathogen effect, which although detectable, did not dominate over the tissue effects. Finally, in Chapter 5, I turned to the crustacean, Daphnia, to study Dscam expression in a natural host-parasite interaction and in a clonal organism. I describe the non-random expression of exons 4 and 6, and another small effect of pathogen-exposure on the expression of Dscam exon 4. My work aimed to further investigate the putative pathogen-specific alternative splicing of the hypervariable Dscam receptor. The data presented quantified the constitutive expression of Dscam exons 4 and 6 in different pancrustacean species. The data also suggest that infection-responsive splicing of Dscam may occur but that effects are small, and may be diluted within the background of the highly important Dscam expression of the nervous system if they exist at all. The study supports the high-throughput sequencing method for future studies of alternative splicing and Dscam expression.
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Spliced leader trans-splicing : a target for the identification of novel anthelmintic drugsPandarakalam, George Cherian January 2016 (has links)
No description available.
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The role of an evolved novel splicing regulatory G tract in diversification of protein functionsSohail, Muhammad 08 September 2015 (has links)
Alternative pre-mRNA splicing greatly contributes to the mammalian proteomic diversity. The novel splice variants often emerge through splicing regulation at/nearby the splice sites (SS). A large group of 3′SS in human genes contain REPA (regulatory elements between the Py and 3′AG) G tracts that mostly appear in mammals as splicing silencers. However, the underlying molecular mechanisms and functional consequences remain unknown.
We have identified a novel class of REPA G tracts (G)5-8 in a group of human genes including PRMT5 (protein arginine methyl transferase 5) that are significantly enriched in functional clusters of cell growth and proliferation. The PRMT5 G tracts emerged evolutionarily in mammals and repress splicing through recruitment of mainly hnRNP H that interferes with early spliceosome assembly. The splicing repression creates a shorter PRMT5 isoform (PRMT5S) that inhibits cell cycle progression contrary to the role of the full length protein (PRMT5L). Moreover, the expression of a group of genes involved in cell cycle arrest at interphase is preferentially regulated by PRMT5S.
We further showed that PRMT5S is differentially expressed among cell and tissue types suggesting tissue-specific regulation. It exhibits distinct subcellular localization pattern from that of PRMT5L and opposite effects on cell cycle-specific structural dynamics of the Golgi apparatus. Moreover, these splice variants are differentially expressed during cell differentiation and PRMT5S promotes the differentiation of dendritic cells whereas PRMT5L shows the opposite effect. The expression of a large number of genes including those involved in crucial cellular processes such as differentiation and apoptosis is regulated by these splice variants of PRMT5.
This study provides a direct link between the evolutionary emergence of a novel splicing regulatory G tract element and the generation of a functionally distinct protein isoform. The molecular mechanism underlying the splicing regulation by this G tract is likely common to many mammalian genes and the generation of their protein diversity. / October 2015
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Cross-species characterisation of alternative splicing patternsTovar-Corona, Jaime M. January 2014 (has links)
Alternative splicing is a common post-transcriptional process in eukaryote organisms by which a single gene can produce more than one distinct transcript. First discovered in the late 1970s, alternative splicing has been the focus of intense attention after the release of the human genome draft revealed a lower than expected gene number. Almost all human protein coding genes are now known to be alternatively spliced. However, how alternative splicing in humans and other well studied model organisms compares to other less characterised taxa such as protists and fungi or what is the functional role of alternative splicing remains poorly understood. Here I analyse alternative splicing in dozens of species using millions of partial transcript sequences ESTs. By applying a transcript normalisation method I showed that alternative splicing in protists and fungi is higher than previously reported and highly variable. I further observed that in representatives of both taxa, associations with translation are overrepresented among alternatively spliced genes. However, no evidence for a relationship between alternative splicing and complex phenotypes was found. Taking human lice as a model I explored the role of alternative splicing in the evolution of phenotypic variants. I found that, despite the fact that the transcriptome profiles of head and body lice are nearly identical, there are markedly differences in alternative splicing patterns. Development related functional associations were found to be enriched among genes with body lice specific alternative splicing events but not in head lice consistent with a scenario of differential patterns of alternative splicing contributing to the phenotypic innovations as human lice adapted to life in human clothing. I further explore the functional relevance of alternative splicing and its possible role in driving genomic innovations even preceding events of gene duplication. Together the work presented show that alternative splicing is widespread among previously understudied fungi and protist species and provide insights on its role in species adaptation to novel environments in using human lice as a model.
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Study into a role for Aar2p in U5 snRNP biogenesisCristao, Vanessa Solange Fernandes de Oliveira January 2011 (has links)
Aar2p is an essential yeast protein involved in pre-mRNA splicing and component of a U5 snRNP precursor form. It has been suggested that the mature U5 snRNP and U4/U6.U5 tri-snRNP assemble from the Aar2p-U5 core particle that is formed in the cytoplasm. After nuclear import, Aar2p would be displaced from its interaction with Prp8p in the Aar2p-U5 particle and the mature U5 snRNP would be formed by interaction of Brr2p with Prp8p. In one model, Aar2p acts as a transport factor either for nuclear import of the Aar2p-U5 particle, or for nucleocytoplasmic shuttling of Prp8p. As a non-mutually exclusive alternative, Aar2p can function as a chaperone, regulating the Prp8p/Brr2p interaction. In this thesis I investigate the role of Aar2p in U5 snRNP biogenesis. I demonstrate by fluorescence microscopy that Aar2p is not required for nuclear localisation of the U5 snRNP components Snu114p and Prp8p. By a yeast two-hybrid (Y2H) assay I establish that Prp8p1649-2413 interacts with Aar2p1-170 but not with Aar2p150-355. I also show for the first time that Aar2p is phosphorylated in five aminoacids: S253, T274, Y328, S331 and T345. In my Y2H system S253 phosphorylation disrupts the Aar2p/Prp8p interaction. This suggests a mechanism whereby formation of the mature U5 snRNP and activation of Brr2p by Prp8p may be regulated through phosphorylation. Surprisingly, when the S253A and S253E mutations are inserted into genomic AAR2 there is no change in the amount of Prp8p and U5 snRNA immunoprecipitated by Aar2p. Finally, using Aar2p1-170 as bait for a Y2H screen, a variety of new Aar2p interactors are revealed. The obtained preys include the other half of Aar2p itself, proteins involved in DNA damage repair, chromatin-binding, ubiquitin-binding and membrane proteins. Overall these results suggest that besides modulating splicing, Aar2p is involved in several other important cellular processes.
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