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I investigated SL trans-splicing in the troponin I gene of Ciona intestinalis. Experimental mutation of the AG dinucleotide adjacent to the natural trans-splice acceptor site (-64) in CiTnI/nuclacZ constructs eliminated trans-splicing to that site in Ciona embryos but activated trans-splicing at cryptic acceptor sites at -76 and -39, adjacent to the nearest AG dinucleotides. However, not all AG dinucleotides specify cryptic acceptor sites because outron internal deletions or 3'truncation mutants were trans-spliced at a far-upstream AG-adjacent cryptic site (-346), leaving many AGs in the retained outron segments. Thus, additional sequence elements that are present only in the -346 and -76/-64/-39 regions are required for cryptic acceptor activity. All mutant constructs generated detectable beta-gal enzyme expression, although the mutant with the longest retained-outron segment appeared less active. Therefore, mRNA accumulation and translation do not require trans-splicing to the natural acceptor site, although they may be facilitated by the normal removal of the outron during trans-splicing.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2008" Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 97-109.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on August 28, 2009). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): William Bosron. Includes vita. Includes bibliographical references (leaves 102-111).

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Splicing mainly occurs co-transcriptionally, suggesting that transcription and premRNA splicing could be synchronized. The nature of this phenomenon suggests that transcription elongation rate may influence splicing outcomes and, indeed, there is evidence for effects on alternative splicing in mammals. To elucidate potential effects of transcription rate on splicing efficiency and fidelity, splicing of nascent transcripts was investigated in fast and slow elongating RNA polymerase II (RNAPII) mutants in Saccharomyces cerevisiae. High kinetic resolution 4-thio Uracil labelling of nascent RNA reveals that fast RNAPII accumulates unspliced pre-mRNA that represents reduced co-transcriptional splicing. Conversely, low levels of unspliced pre-mRNA were detected in the slow mutant due to increased co-transcriptional splicing. The highly stable association of nascent transcripts with elongating RNAPII permits co-transcriptional splicing to be measured by analysis of transcripts that co-purify with RNAPII. Measuring co-precipitation of the spliced mRNA and excised intron that are associated with RNAPII demonstrates that splicing is mostly co-transcriptional with the slow mutant, and the fast mutant reduces co-transcriptional splicing. How elongation rate affects splicing fidelity in budding yeast and whether faster and slower transcription have the opposite effect on splicing fidelity as might be predicted by the kinetic coupling model is an open question. Using deep RNA sequencing, splicing fidelity was determined in yeast transcription elongation mutants. Results show that both fast and slow transcription reduce splicing fidelity mainly in ribosomal protein coding transcripts. Analysis reveals that splicing fidelity depends largely on intron length, secondary structure and splice site score. These analyses also provide new insights regarding the effect of altering transcription rate on selection of transcription start sites. Together, these results indicate that optimal splicing efficiency and fidelity require finely-tuned transcription speed.

The human genome is highly heterogeneous in its GC composition. How codon usage affects translation rates has been extensively studied and exploited to increase protein expression. Although effects on virtually all other steps in gene expression have been reported as well, so far no systematic approach has been taken to quantitatively measure the contribution of each to overall protein levels in human cells. Here, I utilise a library of several hundred synonymous variants of the Green fluorescent protein (GFP) to characterise the influence of codon usage on gene expression in human cells. In an initial small-scale screen, I show that protein levels are largely correlated with codon-usage and particularly GC-content. Additionally, I demonstrate that these changes can already be seen on the RNA level, confirming more broadly previously published data from our lab (Kudla et al., 2006). In order to assess the consequences of randomised codon usage on a larger scale, I established and validated a high-throughput approach for the phenotypic profiling of reporter genes. Using a pool of cells stably expressing >200 GFP variants, I measured multiple parameters simultaneously, such as protein levels, translational state, RNA levels, stability and export. Data from these experiments confirm a strong relationship between GC-content, protein levels, as well as RNA export, reproducibly in two cell lines. Low expression of especially GC-poor variants could not be rescued by splicing, but increased nuclear-to-cytoplasmic RNA ratio, suggesting further mechanisms important for efficient gene expression. These effects are even more pronounced when the distribution of GC is spread evenly along the coding sequence. Interestingly, our data also suggests that high GC within the first 200nt is more predictive of efficient gene expression, contrasting studies performed on bacteria, in which strong secondary folding near the ribosomal binding site was shown to be non-permissive for translation (Kudla et al., 2009). By relating experimentally derived parameters to sequence features known to inhibit expression, I demonstrate that cryptic splicing is a major factor leading to decreased levels of particularly GC-poor GFP variants. An attempt to quantitatively assess the relative contribution of several sequence features (e.g. tAI, GC3, CpG) using multiple regression analysis lead to inconclusive results, leaving the requirement for the exploration of alternative approaches in order to dissect the role of individual parameters, as well as to identify novel determinants of gene expression.

Orientadores: Sérgio Roberto Peres Line, Marcelo Rocha Marques / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T17:45:16Z (GMT). No. of bitstreams: 1 Ribeiro_MarianaMartins_D.pdf: 2903322 bytes, checksum: 9e0e5e91a22262495ca9bf8ae1d84cec (MD5) Previous issue date: 2014 / Resumo: G-Quadruplexes são estruturas secundárias presentes nas moléculas de DNA e RNA, os quais são formados pelo empilhamento de G-quartetos (interação de quatro guaninas (G-tratos) delimitadas por ligações de hidrogênio do tipo Hoogsteen. O intron 1 do gene PAX9 humano tem um G-quadruplex formado na região localizada perto do exon 1, que é conservada entre os mamíferos placentários. Análises de Dicroísmo Circular (CD), e CD melting mostraram que estas sequências são capazes de formar estruturas quadruplex altamente estáveis. Devido à proximidade da estrutura quadruplex ao limite éxon-íntron foi utilizado um ensaio validado de splicing duplo repórter e PCR em tempo real para analisar o seu papel na eficiência de splicing. O quadruplex humano mostrou ter um papel chave na eficiência de splicing do íntron 1 do gene PAX9, já que uma mutação que aboliu a formação do quadruplex diminuiu drasticamente a eficiência de splicing. O quadruplex de rato, menos estável, mostrou menor eficiência quando comparado com sequências humanas. Além disso, o tratamento com 360A, um forte ligante que estabiliza estruturas quadruplex, aumentou ainda mais a eficiência de splicing do íntron 1 do PAX9 humano. Em conjunto estes resultados fornecem evidências de que as estruturas de G-quadruplex estão envolvidas na eficiência de splicing do intron 1 do gene PAX9 / Abstract: G-Quadruplex are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e. interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex- forming region located near exon 1, which is conserved among placental mammals. Using Circular Dichroism (CD) analysis, and CD melting we showed that this region is able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary we used a validated double reporter splicing assay and real time PCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically splicing efficiency. The less stable, rat quadruplex had a less efficient splicing when comparing to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1 / Doutorado / Histologia e Embriologia / Doutora em Biologia Buco-Dental

Orientadores: Jörg Kobarg, José Andrés Yunes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T04:43:04Z (GMT). No. of bitstreams: 1 Righetto_GermannaLima_M.pdf: 4383560 bytes, checksum: b0f7fd3a783153d8832bcf6f44d4b195 (MD5) Previous issue date: 2013 / Resumo: O processo de splicing do RNAm é responsável por orquestrar a junção de exons, criando uma grande diversidade de isoformas gênicas. Alterações nos componentes da maquinaria de splicing e, consequentemente, no processamento do pré-RNAm podem causar ou contribuir para uma infinidade de doenças, dentre elas o câncer. A proteína SF2/ASF foi o primeiro fator de splicing a ser caracterizado como proto-oncogênico, estando superexpresso em diferentes tipos de neoplasias. Sabe-se que a ativação desse fator é, principalmente, mediada por SR quinases conhecidas como splicing quinases e pertencentes à família das SRPKs. A quinase SRPK1, responsável pela fosforilação de SF2/ASF no citoplasma, tem conhecida superexpressão em leucemias. Já a quinase SRPK2, paráloga a SRPK1, possui relação já demonstrada com a proliferação de células leucêmicas e com sua diferenciação. Diante desse quadro, buscamos nesse estudo possíveis correlações entre a maquinaria de splicing e o câncer, dando enfoque à relação entre essa maquinaria e a leucemia. Para tanto, buscamos alterações no cDNA de SRPK2 e quantificamos a expressão das SR quinases SRPK1, SRPK2 e CLK1 em diferentes linhagens de leucemia. Além disso, avaliamos, usando o sistema de Exon Array (Affymetrix), o efeito da superexpressão do fator de splicing SF2/ASF em células de mamífero, buscando alterações globais no splicing dessas células capazes de explicar o caráter oncogênico do fator. Foram encontradas nesse estudo duas novas isoformas de SRPK2, isoladas a partir do cDNA das linhagens de leucemia estudadas. Também foi observada a expressão diferencial das SR quinases SRPK1 e SRPK2 nas linhagens leucêmicas de origem linfóide e mieloide, dando indícios sobre um possível papel divergente dessas quinases nos diferentes tipos de leucemia. Além disso, nas análises preliminares do conjunto de dados obtidos no experimento de Exon Array, foi possível traçar importantes considerações sobre seu caráter oncogênico. Esses dados preliminares do experimento de Exon Array, somados às demais alterações encontradas nas SR quinases, fornecem novas e interessantes pistas sobre a relação entre alterações na maquinaria de splicing e a oncogênese / Abstract: The mRNA splicing is the cellular process responsible for RNA edition, expanding the genome by the combination of gene exons. Mutations in components of this machinery may cause or contribute to a variety of diseases, including cancer. The SF2/ASF protein was the first splicing factor characterized as proto-oncogenic by its overexpression in diverse neoplasias. The cellular activation of this and other splicing factors are mainly dependent on specific kinases, known as splicing kinases and components of a SRPKs family. The SRPK1 kinase, responsible for the cytoplasmic phosphorylation of SF2/ASF, is overexpressed in leukemia. SRPK2, a paralog of SRPK1, is involved in leukemia cell proliferation and differentiation. In this study we searched for splicing machinery and cancer correlations, focusing in the relationship of this machinery and leukemia. In this study we searched for alterations in SRPK2 cDNA and quantified the expression of this kinase and SRPK1 and CLK1 in leukemia immortalized cells. Moreover, we analyzed using Exon Arrays (Affymetrix) the effect of SF2/ASF overexpression in global splicing of non-oncogenic cells, searching for alterations related to its oncogenic character. In this study we discovered two new isoforms of SRPK2 amplified through different leukemia cell lineages. We confirmed the differential expression of SRPK1 and SRPK2 kinases in lymphoid and myeloid leukemia lineages indicating a divergent correlation of these kinases in different leukemia types. In the Exon Array preliminary analysis we also observed important alterations in cellular gene expression. These data and the alterations found in SR kinases provide new and interesting clues about the relationship of splicing machinery alterations and oncogenesis / Mestrado / Genetica Animal e Evolução / Mestra em Genética e Biologia Molecular