Dissertations / Theses on the topic 'RNA splicing'
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Scadden, Alison Deirdre Jane. "Studies of RNA splicing and degradation." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627180.
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Källman, Annika. "Selective ADAR editing and the coordination with splicing /." Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-302.
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Gonzàlez-Porta, Mar. "RNA sequencing for the study of splicing." Thesis, University of Cambridge, 2014. https://www.repository.cam.ac.uk/handle/1810/246596.
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Amongst the many processes that shape the final set of RNA molecules present in eukaryote cells, splicing emerges as the most prominent mechanism for message diversification. In recent years, applications of high throughput sequencing to RNA, known as RNA sequencing, have opened new avenues for the study of transcriptome composition, and have enabled further characterisation of such mechanism. In this thesis, I focus on the application of this technology to the study of human transcript diversity and its potential impact on the protein repertoire. In the first results chapter, I explore the extent of transcriptome diversity by asking whether there is a preference for the production of specific alternative transcripts within each given gene. I show that while many alternative transcripts can be detected, the expression of most protein coding genes tends to be dominated by one single transcript (major transcript). Such findings are validated in the second chapter, and are further used to explore changes in splicing patterns in a disease context. By analysing healthy and tumor samples from kidney cancer patients, I show that most of the detected splicing alterations do not lead to big changes in the relative abundance of major transcripts, at least in a recurrent manner. In addition, I introduce a framework to visualise the most extreme changes in splicing and to evaluate their potential functional impact. In the third chapter, I investigate the role of spliceosome assembly dynamics on the regulation of splice site choice. I show that depletion of PRPF8, a core spliceosomal component, leads to the preferential retention of a subset of introns with weaker splice sites, and also introduces alterations in the rate of co-transcriptional splicing. Finally, in the last chapter, I explore the validation of changes in alternative transcript abundance at the protein level, through the integration of results derived from RNA sequencing datasets with those obtained from proteomics experiments. Altogether, the findings described in this thesis provide a global picture on the extent of alternative splicing in the diversification of the transcriptome, expand current knowledge on the splicing reaction, and open new possibilities for the integration of transcriptomics and proteomics data.
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Friedman, Brad Aaron. "The evolution and specificity of RNA splicing." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37139.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mathematics, 2006.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (p. 119-125).
The majority of human genes are not encoded in contiguous segments in the genome but are rather punctuated by long interruptions known as introns. These introns are copied from generation to generation, and even from cell to cell as a person grows from an embryo into an adult. Each time a gene is activated, the cell must first accurately excise all the introns in a process known as splicing. This excision is determined by the sequence of the gene, but in a complicated way that is not fully understood. By analyzing gene sequences we can learn about how cells decide which sequences to splice. We have developed two new mathematical models, one for the end of introns, and another for long distance interactions between different parts of genes, that expose previously unknown elements potentially involved in the splicing reaction. However their boundaries are determined, introns are very ancient: although they are absent from bacteria they are found in almost all protists, fungi, plants and animals. It is therefore of great interest to explain their evolutionary origins. We have developed a probabilistic model for the evolution of introns and used it to perform a genome-wide analysis of the patterns of intron conservation in four euascomycete fungi, establishing that intron gain and loss are constantly reshaping the distribution of introns in genes.
by Brad Aaron Friedman.
Ph.D.
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Che, Austin 1979. "Engineering RNA logic with synthetic splicing ribozymes." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/47786.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.Includes bibliographical references (p. 169-185).
Reusable components, such as logic gates and code libraries, simplify the design and implementation of electronic circuits and computer programs. The engineering of biological systems would benefit also from reusable components. In this thesis, I show the utility of splicing ribozymes for the biological engineer. Ribozymes allow the engineer to manipulate existing biological systems and to program self-modifying RNA systems. In addition, splicing ribozymes are easy to engineer, malleable, modular, and scalable. I used the model ribozyme from Tetrahymena to explore the principles behind engineering biological splicing systems in vivo. I show that the core ribozyme is modular and functions properly in many different contexts. Simple base pairing rules and computational RNA folding can predict splicing efficiency in bacterial cells. To test our understanding of the ribozyme, I generated synthetic ribozymes by manipulating the primary sequence while maintaining the secondary structure. Results indicate that our biochemical understanding of the ribozyme is accurate enough to support engineering. Splicing ribozymes can form core components in an all-RNA logic system. I developed biological transzystors, switches analogous to electrical transistors. Transzystors can use any trans-RNA as input and any RNA as output, allowing the genetic reading of RNA levels. I also show the ribozyme can write RNA using the trans-splicing reaction.
(cont.) Trans-splicing provides an easy mechanism to hook into an existing biological system and patch its operation. The generality of these ribozymes for a wide set of applications makes them promising tools for synthetic biology. Keywords: synthetic biology, RNA, Tetrahymena, ribozyme, splicing, transzystor.
by Austin J. Che.
Ph.D.
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Robinson, Robert Maxwell. "Splicing signals in Caenorhabditis elegans : candidate exonic splicing enhancer motifs /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/10846.
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Kosmidis, Tilemachos D. "Development of site-specific RNA labeling strategies to probe alternative RNA splicing." Thesis, University of Strathclyde, 2016. http://digitool.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=28492.
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The objective of this project was to develop a method for the labeling of RNA at specific locations using non-natural base pairs. This thesis details the efforts made towards this objective through the development of a modular synthetic platform for an expanded genetic alphabet and its evaluation using transcriptional assays. Chapter 1 introduces the structure and function of RNA, followed by a description of RNA processing events with emphasis placed on alternative RNA splicing and how aberrations in splicing can lead to disease. The concept of RNA labeling follows and examples from the literature are presented along with challenges associated with these techniques to elucidate key mechanistic splicing concepts. The concept of orthogonal base pairs is then introduced along with representative examples from the literature. The application of this concept in RNA labeling is then presented along with key examples from the literature. The limitations of these methods are highlighted followed by the specific aims behind this project. Chapter 2 presents the efforts made towards the development of a modular synthetic route for the synthesis of C-ribonucleosides, with a Heck reaction as the key step. Specifically, its application in the synthesis of the Z ribonucleoside developed by the Benner group is investigated. The chapter starts with re-introducing the Z/P pair developed by the Benner group and highlighting the reasons that placed it as our chosen base pair for RNA labeling. The synthesis of C-deoxyribonucleosides utilizing the Heck reaction is presented, followed by the challenges associated with C-ribonucleoside synthesis. The syntheses of the alkene and halide coupling partners of the proposed Heck reaction are firstly presented. Investigations on the Heck reaction are then presented, followed by investigations on the transformation of nitro group in Z to moieties suitable for further derivatization. The key outcome of this work is that a synthetic route involving the Heck reaction for the synthesis of C-ribonucleosidesis not feasible. This is due to long reaction times required to drive the Heck reaction to completion and difficulties encountered with elucidation of its stereselectivity. Furthermore, PCR experiments conducted by our collaborators within the Eperon group at the University of Leicester revealed significant misincorporation of Z opposite G. Consequently, a change in strategy towards the use of nucleotides developed by the Hirao group was made. Chapter 3 describes the development of a robust synthetic route for the synthesis of a library of C6-functionalized 2-aminopurines as potential candidates for an expanded genetic alphabet in RNA. The s/Pa pair developed by the Hirao group is re-introduced. The installation of an alkyne functionality on the guanosine scaffold via a Sonogashira reaction is described,followed by investigations of [3+2] cycloaddition reactions between the alkyne and azides or aldehyde oximes. Finally, the development of a novel Suzuki-Miyaura protocol for the direct installation of heterocyclic substituents on the guanosine scaffold is also reported. Key outcomes of this chapter are the following. A robust method was developed for the expedient synthesis of C6-functionalized s analogues. This method enabled access to various classes of analogues in three or six steps,including triazoles, isoxazoles, thiophenes and pyrazoles. Attempts to install an azide moiety to the guanosine were partly successful, but the strategy was abanonded due to reaction reproducibility issues. In addition, a C-H activation strategy was not successful on installing an oxazole moiety. Chapter 4 details the efforts towards the synthesis of nucleoside triphosphates based on the s analogues described in Chapter 3. This chapter will begin with the presentation of the most common strategies for the synthesis of nucleoside triphosphates. Initial attempts to synthesize triphosphates by global deprotection of nucleosides synthesized in Chapter 3 are next described. Attempts to install a silyl ether group in the 5’-OH are presented, followed by the installation of acetates on the 2’/3’ hydroxyls and attempts to protect the exocyclic amine on guanosine as the phenoxyacetamide. The installation of a pyrazole moiety via Suzuki-Miyauraprotocol using the corresponding boronic acid pinacol ester described in Chapter 3 is presented. The installation of an alkyne moiety is also described and the synthesis of an isoxazole analogue using this alkyne precursor will follow. The chapter will end with the presentation of the synthesis of pyrazole and isoxazole triphosphate analogues. The key outcome of this chapter is that employing different protecting groups on the 5’ and the 2’/3’ hydroxyls enabled the synthesis of pure triphosphates. Chapter 5 presents the evaluation of the nucleotide triphosphates synthesized in Chapter 4 regarding their transcriptional efficiency. Key outcome of this work are that high concentrations ( > 0.5 mM mM) are needed in order to observe significant incorporation of the analogues, compared to s, which needs 0.1 mM for efficient incorporation. At high concentration, the isoxazole moiety exhibits better incorporation efficiency compared to the pyrazole analogue. Furthermore, the addition of 0.5 mM of MnCl2 resulted in increased incorporation efficiency of the pyrazole analogue, while s and the isoxazole exhibited reduced efficiency under these conditions.
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Hahn, Daniela. "Brr2 RNA helicase and its protein and RNA interactions." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5775.
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The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.
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Weinmeister, Robert. "Development of single-molecule methods for RNA splicing." Thesis, University of Leicester, 2014. http://hdl.handle.net/2381/29311.
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RNA splicing is an important step in the synthesis of most mammalian proteins and understanding the underlying molecular mechanisms is critical for tackling diseases linked to splicing. An important determinant is SRSF1, which has multiple roles in constitutive and alternative splicing. One of these roles is in the recognition and selection of 5’ splice sites due to an interaction with U1 snRNP during formation of complexes E and A. The exact roles and modes of interaction are not clear. Single-molecule methods are a key to understanding these. In this work, single-molecule methods were developed to investigate the number of bound proteins under different conditions. A home-built microscope utilising objective-based illumination by total internal reflection was used to look at these interactions at a single-molecule level and investigate the number of bound proteins under different conditions. The results showed that there was a distinctive reduction in the number of bound proteins in complex A, dependent on the availability of ATP. This was linked to the number of functional 5’ splice sites present, the U1 snRNP and phosphorylation. We could not find any evidence that sequences known to mediate stimulation by SRSF1 affect its binding. Using total internal reflection has inherent limitations, among them the necessary surface attachment and the dilutions required. These limitations could be overcome by the use of isolated microenvironments in the form of tiny droplets. A robust and convenient microfluidic device with a feature size of 3μm was set up and a suitable surfactant for biological samples was identified. Droplets with a diameter of 1μm were generated for the first time using flow focussing and single quantum dots and fluorescent proteins where identified within these droplets. The fluorescence intensity time traces from these droplets enabled the number of encapsulated fluorescent particles to be measured.
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Dickson, Alexa Megan. "Alternative RNA processing and strategies to modulate splicing." Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/6057.
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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.
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Parsons, Aimee. "The relationship between miRNA biogenesis and RNA splicing." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/50970/.
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MiRNAs (miRNAs) are important for the proper regulation of translation, with aberrant miRNA expression contributing to diseases such as cancer. MiRNAs are expressed from longer primary-miRNA transcripts and found in a variety of genomic locations, including introns and exons of coding or long noncoding RNAs. MiRNA biogenesis begins with excision of the precursor miRNA hairpin by the Microprocessor complex which consists of two proteins: Drosha and DGCR8. Both Microprocessor cleavage and splicing occur co-transcriptionally. Splicing and Microprocessing coexist when a miRNA hairpin is located within an intron without detriment to mature mRNA or miRNA production, but little is known about how these two processes interact when a miRNA hairpin is located within an exon. Similarly, little is known about how a miRNA is processed from a long non-coding (lnc)RNA, which is an RNA transcript longer than 200 nucleotides that does not code for a protein. Intronic miRNAs are processed without detriment to the splicing of the host transcript and production of functional mRNA and protein; however, exonic miRNA processing would be expected to lead to cleavage of the exon, interfering with production of mature mRNA. To understand how the genomic location of a miRNA hairpin affects the splicing of the host gene and how splicing affects miRNA production from different locations, two different approaches were used. Firstly, liver-specific miR-122 was inserted at a series of locations within a β-globin plasmid and transfected into HeLa cells, which do not express miR-122. qPCR and northern blotting show that mature miR-122 expression is increased when located within either an intron or exon compared to its endogenous context within a long noncoding RNA. Importantly, the levels of spliced β-globin was shown to decrease when miR-122 was expressed from an exon. This suggests that there is some competition between miRNA biogenesis and splicing. Secondly, the effect of the splice inhibitor pla B was used to understand the effect of splicing on endogenous pri-miR-122. Splice inhibition led to a decrease in the levels of spliced pri-miR-122, but interestingly also led to a decrease in the levels of unspliced transcript indicating that splice inhibition strongly reduces the transcription of pri-miR-122. This was found to be unique to the endogenous gene, as it was not seen when pri-miR-122 was ectopically expressed in HeLa cells. Analysis of the chromatin-associated RNA further confirmed that splice inhibition was having a strong negative effect on transcription. Transcription of pri-miR-122 is known to be terminated by Microprocessing rather than the canonical pathway, and analysis of other lnc-pri-miRNAs which are Microprocessor terminated showed that the effect of splice inhibition on transcription was not unique to pri-miR-122, but was also shared by the important oncogene pri-miR-17~92a. The results in this thesis show that intronic or exonic miRNA hairpins do have an effect on splicing, and unexpectedly that splicing is important for efficient transcription of specific lnc-pri-miRNAs. This work shows that the relationship between Microprocessing and splicing, as well as transcription, is complex. By elucidating how these co-transcriptional processes occur on the same transcript, the understanding of how miRNA biogenesis is regulated from different genomic locations will be improved. This could further the current knowledge of how miRNA disregulation occurs in disease and may lead to the development of new drugs to treat them.
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Mitrpant, Chalermchai. "Pre-mRNA splicing manipulation via Antisense Oligomers." Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2009. https://ro.ecu.edu.au/theses/421.
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Duchenne muscular dystrophy (DMD), the most common lethal neuromuscular disease in childhood, arises from protein-truncating mutations in the dystrophin gene. A deficiency in dystrophin leads to loss of the dystrophin associated protein complex (DAPC), which in turn, renders muscle fibres vulnerable to injury, and eventually leads to muscle loss, necrosis and fibrosis. Although, the dystrophin gene was identified nearly two decades ago, and extensive research has been directed at finding a therapy for DMD, to date, there is still no effective treatment available. One promising molecular approach to treat DNID is antisense oligomer (AO) induced splice intervention. AOs were most widely used to induce RNaseH-mediated gene transcript degradation, however, the development of different backbone chemistries heralds a new generation of AOs that can modify gene transcript splicing patterns. Application of AOs to the dystrophin pre-mRNA to influence exon selection and induce shortened, in-frame dystrophin isoforms is being vigorously pursued. The majority of the work presented here explores the concept of personalised therapies for DMD, whereby oligomers are designed to specifically target individual mutations. The importance of AO-optimisation to obtain AOs capable of inducing efficient dual exon skipping in an established animal model of muscular dystrophy (4CV mouse), which carries a DMD-causing mutation in exon 53, is demonstrated. Removal of both exons 52 and 53 was required to by-pass the mutation, maintain the reading frame and restore dystrophin expression. One of the major challenges of AO-induced splice intervention for therapeutic purposes will be the design and development of clinically relevant oligomers for many different mutations. Various models, including cells transfected with artificial constructs and mice carrying a human dystrophin transgene, have been proposed as tools to facilitate oligomer design for splice manipulation. This thesis investigates the relevance of using mouse models to design AOs for human application, and also explores the use of cultured human myoblasts, from both unaffected humans and a DMD patient, as a means of establishing the most effective therapeutic compound. In addition to induction of exon skipping, the applicability of AOs to promote exon inclusion, by masking possible intronic silencing motifs of survival motor neuron (SMN) pre-mRNA in cultured fibroblasts from a spinal muscular atrophy (SMA) patient, is investigated. This study provides additional information about a novel oligomer target site that could be used in combination with previously identified splice silencing motifs for a molecular therapeutic approach to SMA, and may perhaps open up new avenues of treatment for other genetic disorders, where oIigomers could be used to induce exon inclusion.
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Wang, Weizhong. "Nuclear galectins and their role in pre-mRNA splicing." Diss., Connect to online resource - MSU authorized users, 2006.
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Thesis (Ph. D.)--Michigan State University. Dept. of Microbiology and Molecular Genetics, 2006.Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.
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Harris, Lorena B. ""Characterization of a small ribozyme with self-splicing activity"." Bowling Green, Ohio : Bowling Green State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=bgsu1225668492.
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DI, MATTEO ANNA. "An RNA map of Nova2-regulated alternative splicing in endothelial cells." Doctoral thesis, Università degli studi di Pavia, 2017. http://hdl.handle.net/11571/1203363.
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Lo splicing alternativo (AS) genera diversi mRNAs che portano alla produzione di isoforme proteiche con diverse proprietà funzionali. Poiché più del 90% dei geni umani subisce AS, esso svolge un ruolo importante nell’aumentare la capacità codificante del nostro genoma. E’ stato ampiamente dimostrato che AS è fondamentale per regolare numerosi processi cellularI; inoltre una sua deregolazione ha un ruolo chiave nella progressione tumorale. L’alterazione di AS che si osserva nei tumori è spesso causata da cambiamenti nei livelli di espressione di fattori di splicing; questo porta alla inattivazione di oncosoppressori e all’attivazione di oncoproteine coinvolte nello sviluppo e progressione della neoplasia, nonché nella resistenza ai trattamenti terapeutici.
L'angiogenesi, la crescita di nuovi vasi sanguigni da vasi preesistenti, svolge un ruolo cruciale nello sviluppo del tumore, consentendo all’ossigeno e alle sostanze nutritive di raggiungere le cellule tumo-rali proliferanti. Negli ultimi anni, il processo di angiogenesi è stata molto studiato poichè la sua inibizione rappresenta una promettente strategia terapeutica contro il cancro. Tuttavia, tali terapie fino ad oggi hanno mostrato solo modesti effetti terapeutici indicando che l'angiogenesi tumorale è un fenomeno molto più complesso di quan-to precedentemente previsto. Quindi, una migliore comprensione dei meccanismi molecolari che sostengono la crescita dei vasi tumorali sarà fondamentale per individuare nuove e più efficaci terapie anti-angiogenetiche per il trattamento del cancro.
Fino ad ora i meccanismi molecolari coinvolti nell’angiogenesi so-no stati principalmente investigati a livello della regolazione genica trascrizionale. Recentemente però l’importanza dei meccanismi di regolazione genica post-trascrizionale durante lo sviluppo vascolare è stata rivalutata.
Per la prima volta, il nostro gruppo ha dimostrato che la formazione del sistema vascolare è controllata a livello post-trascrizionale dal fattore di AS Nova2, precedentemente considerato espresso solo dalle cellule neuronali. Attraverso la regolazione di eventi di AS di geni codificanti per membri del complesso Par e per sue proteine regolatrici, Nova2 controlla l’acquisizione della polarità da parte della cellule endoteliali (ECs) e l’organizzazione del lume vascolare durante l’angiogenesi. L’assenza di Nova2 in vivo causa difetti nella formazione del lume vascolare e questo fenotipo ricorda, sotto alcuni aspetti, la morfologia aberrante della vascolatura tumorale.
Utilizzando il sequenziamento massivo dell’RNA estratto da cellule ECs deplete o overesprimenti Nova2, ho identificato nuovi esoni regolati mediante AS da Nova2 in endotelio; molti di questi esoni appartengono a geni coinvolti nell’angiogenesi o nello sviluppo vascolare, suggerendo che Nova2 ha un ruolo fondamentale nel regolare la biologia delle ECs che compongono i vasi sanguigni.
Tra i nuovi bersagli di Nova2 che ho identificato c’è Ptbp2, un altro fattore di AS la cui espressione era stata fino ad ora considerata ristretta solo al cervello e al testicolo. Ho dimostrato che Nova2 reprime l’espressione di Ptbp2 nelle ECs promuovendo l’esclusione dell’esone 10 e la produzione di un trascritto di Ptbp2 che viene degradato attraverso il processo di NMD (“Non-sense Mediated mRNA Decay”). Ho trovato che la regolazione di Ptbp2 è conservata specificatamente nell’endotelio del pesce zebra. I risultati da me ottenuti rivelano una gerarchia di fattori di splicing che integrano le decisioni di splicing alternativo durante l’angiogenesi.Alternative splicing (AS) generates different mRNAs leading to production of protein isoforms with different functional properties. Since more than 90% of the human genes undergo AS, it plays a major role in the generation of human proteomic diversity. There is now ample evidence that just as AS is important for normal physiology, so altered AS is important for cancer. Altered AS in tumors is frequently due to changes in the levels of AS regulators leading to combined disruption of tumor suppressor genes and activation of oncoproteins involved in tumor establishment, progression and in resistance to therapeutic treatments. Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays a crucial role in tumor development by allowing oxygen and nutrients to reach proliferating cancer cells. In recent years, angiogenesis has been deeply investigated because its inhibition represents a promising anti-cancer therapeutic modality. However, all attempted strategies to date have shown modest therapeutic effects indicating that tumor angiogenesis is a more complex phenomenon than previously anticipated. Hence, a better understanding of the mechanisms sustaining growth of tumor vessels will be crucial to identify novel and effective anti-angiogenic therapies for cancer treatment. Traditionally, the molecular pathways involved in angiogenesis have been suggested to act primarily through regulation of transcription. Recently, the emphasis on crucial events of gene expression is changing, as many post-transcriptional programs cooperate to promote vascular development. For the first time, our group found that the formation of vascular lumen during angiogenesis is controlled at post-transcriptional level by the AS factor Nova2, previously considered neural cell-specific. Through AS of target exons affecting the Par complex and its regulators, Nova2 controls the establishment of endothelial cell polarity, a prerequisite for vascular lumen organization. Consequently, Nova2 in vivo ablation causes vascular lumen formation defects, reminiscent of aberrant morphology of the tumor vasculature. By performing RNA-seq of Nova2 depleted or overexpressing endothelial cells (ECs), I identified novel Nova2-mediated AS exons belonging to factors involved in angiogenesis or vascular development suggesting that Nova2 controls an essential layer of vascular gene expression regulation. Among the novel identified Nova2 targets in ECs there is Ptbp2, an AS factor until now considered restricted to brain and testis. Notably, I demonstrated that Nova2 functions to repress Ptbp2 expression in ECs by promoting skipping of Ptbp2 exon 10 leading to the production of a Ptbp2 transcript degraded by the NMD (Non-sense Mediated mRNA Decay) pathway. Importantly, I found that Nova2 dependent AS regulation of Ptbp2 is specifically conserved in zebrafish endothelium. Collectively, my results reveal a hierarchy of splicing factors that integrate splicing decisions during angiogenesis.
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Wetterberg, Ingela. "Biochemical and structural studies of pre-mRNA splicing /." Stockholm : Karolinska Univ. Press, 2001. http://diss.kib.ki.se/2001/91-7349-017-2/.
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Cass, Danielle Marie. "Role of two RNA binding properties in pre-mRNA splicing /." view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1400950811&sid=2&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-80). Also available for download via the World Wide Web; free to University of Oregon users.
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Hage, Rosemary. "Identification and characterization of YNL187, a novel factor that promotes stable association of the U1 SNRNP with the 5’SS during pre-messenger RNA splicing." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1193268127.
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Hannon, Gregory James. "Trans-splicing of nematode pre-messenger RNA." Case Western Reserve University School of Graduate Studies / OhioLINK, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=case1059657008.
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Yu, Yi-Tao. "SL RNA and U6snRNA sequence requirements for nematodetrans-splicing." Case Western Reserve University School of Graduate Studies / OhioLINK, 1994. http://rave.ohiolink.edu/etdc/view?acc_num=case1061564747.
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Vu, Ngoc T. "Regulation and Mechanistic Functions of Caspase-9 RNA Splicing." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3578.
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Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.
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Reid, Jane Elizabeth Anne. "An 'AID' to understanding links between splicing and transcription." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17929.
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This study seeks to address one of the simplest questions that can be asked about an interconnected system; what happens to one process in the absence of the other process? This is a more difficult task than it would appear at first, due to the absence of small molecule inhibitors that can inhibit splicing globally in yeast cells. The first results chapter describes the adaptation of a system called the auxin induced degron (AID) to the task of inhibiting pre-mRNA splicing. This system appears to have several advantages over previous methods of inhibiting splicing and has many potential applications. Another hurdle to understanding what happens to transcription in the absence of splicing is the differential stability of pre-mRNA versus mRNA. At steady state the vast majority of transcripts of a specified gene will be mRNA transcripts. This means that even if you could rapidly inhibit splicing it would be a long time before all the pre-existing mRNA would turn-over. If you waited until specified mRNAs turned over it is likely that the cells would be very sick making it difficult to separate primary and secondary effects. The second results chapter shows the use of a metabolic labelling technique using a uracil analogue called 4-thiouracil (4SU). 4SU is added for an extremely short amount of time (1.5 min, 2.5 min, and 5 min) and the RNA produced during the labelling time is isolated by affinity purification. This allows us to study the kinetics of pre-mRNA splicing in wild-type cells and to seek correlations between splicing kinetics and gene architecture. The third results chapter combines the methods used in the previous two chapters to give a new technique called AID4U-seq. AID4U-seq allows for rapid inhibition of splicing and then the ability to isolate only the transcripts that were created after this inhibition came into effect. This should allow for examination of the primary consequences of blocking pre-mRNA splicing at multiple stages during spliceosome assembly. Additionally AID4U-seq is immediately applicable to the study of other areas of RNA processing. Defining the effects on the transcriptome of inhibiting splicing at multiple stages of assembly is an ambitious aim likely to require many more years of research. Therefore this thesis chiefly seeks to illustrate a novel strategy to begin dissecting a complex issue in which splicing, transcription, degradation and the post-transcriptional modification of histones are all likely to have roles.
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Deyoung, Katherine Leigh. "Genetic studies of self-splicing RNAs in bacteriophage T4." Diss., Georgia Institute of Technology, 1993. http://hdl.handle.net/1853/25434.
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Long, Jennifer Connie. "Identification of in vivo RNA tragets of the RNA-binding proteins Acinus and hnRNP A1." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4227.
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RNA-binding proteins play a central role in the post-transcriptional regulation of gene expression; however, little is known about the endogenous transcripts to which they bind. Here, I have used the ultra-violet cross-linking and immuno-precipitation (CLIP) technique to identify RNA targets directly bound to two RNA-binding proteins: Acinus and hnRNP A1. Acinus (apoptotic chromatin condensation inducer in the nucleus) contains a region that is homologous to the RNA binding domain of the Drosophila splicing regulator sex-lethal, and a serine and arginine rich region similar to that seen in the SR family of proteins, which function extensively in splicing. Furthermore it is a component of the multi-protein spliceosome complex, and I have demonstrated it can directly bind polyadenylated RNA. I have shown that Acinus displays a diffuse nuclear localisation pattern, however, overexpression of an epitope-tagged protein results in its accumulation in enlarged nuclear speckles. Together these results suggest a role in pre-mRNA splicing. Acinus is cleaved during apoptosis by caspase-3, resulting in a truncated protein with chromatin condensation inducing activity (Sahara et al., 1999). Accordingly, I have demonstrated that overexpression of epitope-tagged Acinus results in an increased number of cells exhibiting an apoptotic phenotype. The proteolytic fragment contains the RNA binding region, and to determine if the role of Acinus in apoptosis is mediated by RNA interactions I utilised CLIP to identify in vivo RNA targets. I have identified several mRNA targets of Acinus and found that the binding sites in those mRNA targets predominantly map to constitutively expressed exons. This is in agreement with the exon junction complex, of which Acinus is a component, being deposited on mRNAs after splicing. These results may indicate that Acinus is a core RNA binding factor of the exon junction complex. To complement this approach, I also performed CLIP with a known alternative splicing regulator, hnRNP A1. In this manner, the binding site preferences could be compared between the two proteins. As expected, the majority of hnRNP A1 binding sites are located in introns, corresponding with their identified role of antagonizing pre-mRNA splicing by binding intronic splicing elements. Interestingly, a number of the CLIP tags are located in, or adjacent to, alternatively spliced events suggesting a role for hnRNP A1 in the regulation of alternative splicing of these specific pre-mRNAs. In addition to pre-mRNA splicing hnRNP A1 also functions in the cellular stress response. Upon environmental stresses it relocates to the cytoplasm and accumulates in cytoplasmic foci known as stress granules (Guil et al., 2006). Here I show some of the targets identified by CLIP are regulated by hnRNP A1 in times of cellular stress. In summary, I have identified two novel subsets of RNAs, bound by Acinus or hnRNP A1 in vivo. I have shown these proteins exhibit distinct binding preferences, which correspond to their biological function. This work is consistent with hnRNP A1 acting as an alternative splicing regulator, and provides evidence for a dual role of Acinus in mRNA splicing and apoptosis. This study also demonstrates the power of the CLIP technique, as identification of in vivo RNA targets allows greater understanding of the mechanisms by which RNA-binding proteins exert their regulatory control.
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Brown, Michael Dean. "Genetic analysis of RNA splicing in the thymidylate synthase gene of bacteriophage T4." Diss., Georgia Institute of Technology, 1990. http://hdl.handle.net/1853/25390.
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Stands, Leah Rae. "Regulation of U1 snRNP / 5' splice site interactions during pre-MRNA splicing in Saccharomyces cerevisiae." Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1056395043.
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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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Murray, Jill Isobel. "Identification of motifs that function in the splicing of non-canonical introns /." Connect to title online (ProQuest), 2007. http://proquest.umi.com/pqdweb?did=1453227351&sid=1&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 76-84). Also available online in ProQuest, free to University of Oregon users.
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Lal, Sunil Kumar. "Genetic studies of RNA splicing in the ribonucleotide reductase small subunit of bacteriophage T4." Diss., Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/30994.
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Jarrell, Kevin A. "Cis and trans reactions of a self-splicing group II intron /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487584612166082.
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Dotson, Perry Patrick II. "MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION-SPLICING AND TRANS INSERTION-SPLICING REACTION." UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/605.
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Group I intron-derived ribozymes are catalytic RNAs that have been engineered to catalyze a variety of different reactions, in addition to the native self-splicing reaction. One such ribozyme, derived from a group I intron of Pneumocystis carinii, can modify RNA transcripts through either the excision or insertion of RNA sequences. These reactions are mediated through the trans excision-splicing (TES) or trans insertionsplicing (TIS) reaction pathways. To increase our current understanding of these reactions, as well as their general applicability, a mechanistic and kinetic framework for the TES reaction was established. Furthermore, additional ribozymes were investigated for their ability to catalyze the TES reaction. Lastly, the development of the TIS reaction into a viable strategy for the manipulation of RNA transcripts was investigated.
The TES reaction proceeds through two reaction steps: substrate cleavage followed by exon ligation. Mechanistic studies revealed that substrate cleavage is catalyzed by the 3’ terminal guanosine of the Pneumocystis ribozyme. Moreover, kinetic studies suggest that a conformational change exists between the individual reaction steps. Intron-derived ribozymes from Tetrahymena thermophila and Candida albicans were also investigated for their propensity to catalyze the TES reaction. The results showed that each ribozyme could catalyze the TES reaction; however, Pneumocystis carinii is the most effective using the model constructs.
Investigations of the TIS reaction focused on developing a new strategy for the insertion of modified oligonucleotides into an RNA substrate. These studies used oligonucleotides with modifications to the sugar, base, and backbone positions. Each of the modified oligonucleotides was shown to be an effective TIS substrate. These results demonstrate that TIS is a viable strategy for the incorporation of modified oligonucleotides, of varying composition, into an intended RNA target.
The results from these studies show that group I introns are highly adaptable for catalyzing non-native reactions, including the TES and TIS reactions. Furthermore, group I introns are capable of catalyzing these unique reactions through distinct reaction pathways. Overall, these results demonstrate that group I introns are multi-faceted catalysts.
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Khan, Dilshad Hussain. "Role of histone deacetylases in gene expression and RNA splicing." Informa UK Limited, 2012. http://hdl.handle.net/1993/22163.
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Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression.
During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes.
We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism.
Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.
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Huang, Yuanhua. "Structured Bayesian methods for splicing analysis in RNA-seq data." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31328.
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In most eukaryotes, alternative splicing is an important regulatory mechanism of gene expression that results in a single gene coding for multiple protein isoforms, thus largely increases the diversity of the proteome. RNA-seq is widely used for genome-wide splicing isoform quantification, and several effective and powerful methods have been developed for splicing analysis with RNA-seq data. However, it remains problematic for genes with low coverages or large number of isoforms. These difficulties may in principle be ameliorated by exploiting correlations encoded in the structured data sources. This thesis contributes to developments of Bayesian methods for splicing analysis by leveraging additional information in multiple datasets with structured prior distributions. First, we developed DICEseq, the first isoform quantification method tailored to time-series RNA-seq experiments. DICEseq explicitly models the correlations between experiments at different time points to aid the quantification of isoforms across experiments. Numerical experiments on both simulated and real datasets show that DICEseq yields more accurate results than state-of-the-art methods, an advantage that can become considerable at low coverage levels. Furthermore, DICEseq permits to quantify the trade-off between temporal sampling of RNA and depth of sequencing, frequently an important choice when planning experiments. Second, we developed BRIE (Bayesian Regression for Isoform Estimation), a Bayesian hierarchical model which resolves the difficulties in splicing analysis in single-cell RNA-seq (scRNA-seq) data by learning an informative prior distribution from sequence features. This method combines the quantification and imputation for splicing analysis via a Bayesian way, which is particularly useful in scRNA-seq data due to its extreme low coverages and high technical noises. We validated BRIE on several scRNA-seq data sets, showing that BRIE yields reproducible estimates of exon inclusion ratios in single cells. Third, we provided an effective tool by using Bayes factor to sensitively detect differential splicing between different single cells. When applying BRIE to a few real datasets, we found interesting heterogeneity patterns in splicing events across cell population, for example alternative exons in DNMT3B. In summary, this thesis proposes structured Bayesian methods to integrate multiple datasets to improve splicing analysis and study its biological functions.
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Aslanzadeh, Vahid. "Tuning the RNAPII elongation rate is required for optimal pre-mRNA splicing efficiency and fidelity." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/29593.
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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.
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Henscheid, Kristy L. "Functional conservation and RNA binding of the pre-mRNA splicing factor U2AF65 /." view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1400950821&sid=5&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 129-141). Also available for download via the World Wide Web; free to University of Oregon users.
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Kolisnichenko, Marina. "The role of the polyadenylation site of the melanocortin 1 receptor in generating MC1R-TUBB3 chimeras and attenuation of TORC1 delays the onset of replicative and RAS-induced cellular senescience." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711654.
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Boralli, Camila Maria dos Santos. "Estudo do envolvimento da RNA helicase Sub2 na reação de splicing em Trypanosoma brucei." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-03102018-092702/.
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A excisão de sequencias intrônicas dos precursores de mRNAs é um passo crítico durante a expressão gênica eucariótica. Essa reação é catalisada pelo complexo macromolecular denominado spliceossomo, composto por partículas ribonucleoproteicas nucleares (U1, U2, U4/U6, U5 snRNPs), além de inúmeros fatores associados. Em tripanossomatídeos, os genes são transcritos em longas unidades policistrônicas e a reação de SL trans-splicing é requerida para geração de transcritos monocistrônicos maduros. O spliceossomo é uma maquinaria altamente dinâmica e parte das mudanças conformacionais ocorridas neste complexo é mediada por RNA helicases. A RNA helicase/ATPásica Sub2 (em mamífero, UAP56), cujas homólogas foram descritas como membros do complexo TREX (transcrição/exportação do RNA), é essencial na montagem do pré-spliceossomo, além de estudos sugerirem sua participação em outras etapas de montagem desse complexo. Em tripanossomatídeos, a função desta proteína no transporte de mRNA entre núcleo e citoplasma já foi descrita, entretanto, seu envolvimento na reação de splicing permanece indefinido. Neste trabalho, buscou-se estudar esse envolvimento através de técnicas de purificação em tandem e RNA de interferência. A purificação dos parceiros de interação da proteína somada à identificação por espectrometria de massas mostrou que TbSub2 co-purifica com proteínas envolvidas em múltiplas vias do metabolismo do parasito, incluindo proteínas e fatores relacionados ao processamento de mRNA. Além disso, essa proteína é essencial para os parasitos na forma procíclica e sanguínea e apresenta localização nuclear em ambas as linhagens. Análises por qPCR em tempo real e RT-PCR mostraram que o silenciamento de TbSub2 causa defeito na maquinaria de SL trans-splicing mas seu efeito no cis-splicing não é claro. Por fim, foi possível realizar a expressão heteróloga e purificação da proteína TbSub2 recombinante, bem como estudos para avaliar seu estado oligomérico e estabilidade. Dessa forma, foi constatado que essa proteína é estável em diferentes tampões e apresenta estados oligoméricos distintos nas técnicas empregadas nesse trabalho. O estudo aqui apresentado trouxe evidências da participação da proteína TbSub2 na reação de SL trans-splicing em ambas as linhagens, bem como em múltiplas vias do metabolismo do parasito na forma procíclica, contribuindo para uma elucidação das funções dessa proteína em tripanossomatídeos.The excision of intronic sequences from precursor mRNAs is a critical step during eukaryotic gene expression. This reaction is catalyzed by the macromolecular complex called spliceosome, composed of nuclear ribonucleoprotein particles (U1, U2, U4 / U6, U5 snRNPs), besides numerous associated factors. In trypanosomatids, genes are transcribed in long polycistronic units and the SL trans-splicing reaction is required for the generation of mature monocistronic transcripts. The spliceosome is a highly dynamic machinery and part of this complex\'s conformational changes is regulated by RNA helicases. The RNA helicase / ATPase Sub2 UAP56, in mammalian), whose homologous proteins have been described as members of the TREX complex (transcription / RNA export), is essential in the pre-spliceosome assembly, besides its participation in other assembly steps of this complex, as suggested by other studies. In trypanosomatids, the role of this protein in the nucleus/cytoplasm mRNA transport has already been described, however its involvement in the splicing reaction remains unknown. In this work, we studied this involvement through tandem affinity purification and RNA interference. The purification of this proteins binding partners added to the identification by mass spectrometry showed that TbSub2 co-purifies with proteins involved in multiple metabolism pathways of the parasite, including proteins and factors related to mRNA processing. In addition, this protein is essencial for the parasites in procyclic and bloodstream forms and localizes in the nucleus in both strains. qPCR and RT-PCR analysis showed that the silencing of TbSub2 causes defect in the SL trans-splicing machinery but its effect on cis-splicing is unclear. Finally, it was possible to perform the heterologous expression and purification of recombinant TbSub2 protein, as well as studies to evaluate its oligomeric state and stability. Thus, it was found that this protein is stable in different buffers and presents different oligomeric states in the techniques employed in this work. This study has provided evidence of this helicases participation in the SL trans-splicing reaction in booth strains, as well as in several metabolism pathways of the parasite in prociclic form, contributing to the elucidation of the protein functions in trypanosomatids.
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Stark, Jeremy M. "SR proteins can function during alternative splicing to mediate exon/exon associations /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5020.
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Zaghlool, Ammar. "Genome-wide Characterization of RNA Expression and Processing." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-209390.
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The production of fully mature protein-coding transcripts is an intricate process that involves numerous regulation steps. The complexity of these steps provides the means for multilayered control of gene expression. Comprehensive understanding of gene expression regulation is essential for interpreting the role of gene expression programs in tissue specificity, development and disease. In this thesis, we aim to provide a better global view of the human transcriptome, focusing on its content, synthesis, processing and regulation using next-generation sequencing as a read-out. In Paper I, we show that sequencing of total RNA provides unique insights into RNA processing. Our results revealed that co-transcriptional splicing is a widespread mechanism in human and chimpanzee brain tissues. We also found a correlation between slowly removed introns and alternative splicing. In Paper II, we explore the benefits of exome capture approaches in combination with RNA-sequencing to detect transcripts expressed at low-levels. Based on our results, we demonstrate that this approach increases the sensitivity for detecting low level transcripts and leads to the identification of novel exons and splice isoforms. In Paper III, we highlight the advantages of performing RNA-sequencing on separate cytoplasmic and nuclear RNA fractions. In comparison with conventional poly(A) RNA, cytoplasmic RNA contained a significantly higher fraction of exonic sequence, providing increased sensitivity for splice junction detection and for improved de novo assembly. Conversely, the nuclear fraction showed an enrichment of unprocessed RNA compared to when sequencing total RNA, making it suitable for analysis of RNA processing dynamics. In Paper IV, we used exome sequencing to sequence the DNA of a patient with unexplained intellectual disability and identified a de novo mutation in BAZ1A, which encodes the chromatin-remodeling factor ACF1. Functional studies indicated that the mutation influences the expression of genes involved in extracellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlated with the patient’s clinical diagnosis. The findings presented in this thesis contribute towards an improved understanding of the human transcriptome in health and disease, and highlight the advantages of developing novel methods to obtain global and comprehensive views of the transcriptome.
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Savas, Isabella. "Alternativ splicing: en process som medför att flera olika mRNA-transkript bildas från individuella gener." Thesis, Linköping University, Department of Physics, Chemistry and Biology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-56880.
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This review article presents the splicing process during messenger RNA maturation and how it is regulated by different Cis-regulatory RNA-sequence elements and splicing factors. A more detailed description of the process alternative splicing and its importance to the function of genes from the model organism Arabidopsis thaliana is also given. A single eukaryotic gene can by the process alternative splicing (AS) give rise to a number of functionally mature mRNA-molecules, which in turn encodes for structurally and/or functionally different proteins. During the course of evolution, the process alternative splicing has thus shown to be effective in increasing transcriptome and proteome diversity of most eukaryotic organisms. This suggests therefore that the dominant theory in molecular biology, a gene encodes for a protein, needs to be corrected. A future challenge is to determine the function of the proteins obtained from a given gene by alternative splicing.
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Garrey, Stephen M. "Characterization of the specificity and affinity of the splicing factor BBP/SF1 /." view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1324375731&sid=2&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 81-88). Also available for download via the World Wide Web; free to University of Oregon users.
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Keegan, Niall Patrick. "Hidden stitches: RNA cryptic splicing and its role in human disease." Thesis, Keegan, Niall Patrick (2022) Hidden stitches: RNA cryptic splicing and its role in human disease. PhD thesis, Murdoch University, 2022. https://researchrepository.murdoch.edu.au/id/eprint/64691/.
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A great majority of human genes contain introns: tracts of mostly non-functional sequence that intervene the functional exons. When intron-bearing genes are transcribed into RNA, the introns are removed from the transcript via splicing, a process controlled by a multimolecular assembly called the spliceosome.
Although splicing is generally well-regulated, the spliceosome sometimes splices RNA transcripts at sites other than their canonical exon boundaries. This “cryptic” splicing can be a random event, part of an unidentified regulatory process, the effect of a mutation, or the result of other perturbances to the spliceosome’s normal behaviour.
In this thesis, I present four reports on the mechanisms underlying certain forms of cryptic splicing. In the first report, an analysis of pathogenic pseudoexons in the DMD gene reveals that each causative mutation falls into a distinct category defined by its proximity to the pseudoexon, and that many DMD pseudoexon splice sites are actively spliced in non-mutant cells. The second report builds on this by constructing a catalogue of over 400 pseudoexon variants from across the human transcriptome and uses this dataset to propose new and revised pseudoexon mutation categories. Like the first report, this second report also finds substantial congruence between pseudoexons and active deep intronic splice sites – including several recursive splice sites – suggesting a causal link between these phenomena.
A third report explores how some cryptic exons may provide an explanatory mechanism to connect common genetic variants with their associated population phenotypes and outlines a simple method for discovering new examples.
The fourth and final report uses RNA secondary structure modelling to explain why some antisense oligonucleotides can induce partial exon skipping through cryptic splice-site activation.
Collectively, these reports present several novel insights into the causes of cryptic splicing and offer suggestions for how future research may build upon these insights.
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Pandarakalam, George Cherian. "Spliced leader trans-splicing : a target for the identification of novel anthelmintic drugs." Thesis, University of Aberdeen, 2016. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=230533.
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Ostheimer, Gerard Joseph. "Proteins required for chloroplast group II intron splicing : CRS2 and its associated factors /." view abstract or download file of text, 2003. http://wwwlib.umi.com/cr/uoregon/fullcit?p3080594.
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Thesis (Ph. D.)--University of Oregon, 2003.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 111-124). Also available for download via the World Wide Web; free to University of Oregon users.
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Huang, Ching-jung. "The role of HnRNP proteins, PSF and nonO/p54[superscript nrb], in pre-mRNA binding and splicing /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004292.
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Ribeiro, Mariana Martins 1984. "G-quadruplex formation enhances splicing efficiency of PAX9 intron 1." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290066.
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Orientadores: Sérgio Roberto Peres Line, Marcelo Rocha MarquesTexto em português e inglês
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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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
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Paiva, Marcelo Machado. "Identificação de proteínas reguladoras do splicing associadas à microRNAs." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-10112016-101050/.
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Splicing é o processo de remoção de introns e ligação de exons em eucariotos. É realizado pelo spliceossomo, um complexo macromolecular composto por RNAs e mais de cem proteínas. Alguns introns possuem microRNAs, os quais devem ser processados para gerar moléculas maduras. O cluster intrônico miR-17-92 é composto por sete miRNAs que têm sido associados ao desenvolvimento de diferentes tumores em vários tecidos. Neste trabalho o splicing de dois miRNAs deste cluster foi analisado em células HeLa, BCPAP e TPC-I. Os resultados mostraram que introns com miR19a tem o splicing mais eficiente do que aqueles com miR18a em todas as três células analisadas. Além disso, a composição dos spliceossomos foi analisada por espectrometria de massas. Entre as principais proteínas encontradas, destaca-se a presença das hnRNPs, como hnRNP_A1 e hnRNP_A2/B1. Estes resultados são importantes para entender como esses miRNAs são processados, e quais são os principais componentes recrutados em diferentes tipos celulares.Pre-mRNA splicing is the process of intron removal and exon ligation in eukaryotes. It is performed by the spliceosome, a multi-megadalton machinery composed of RNAs and more than a hundred proteins. Intronic miRNAs must be processed from the host gene to generate mature molecules. miR-17-92 is an intronic cluster composed of seven miRNAs which have been associated to the development of different tumors, in several different cells. In this work, we analyzed the splicing of two miRNAs belonging to this cluster in HeLa, BCPAP and TPC-I cells. Interestingly, we observed miR19a is more efficiently spliced than miR18a in all three cells. We also searched for specific proteins that can be involved in their respective splicing process. We observed hnRNP proteins are especially concentrated in spliceosomes assembled in introns containing these miRNAs, based on mass spectrometry data. These results are important to understand how these miRNAs are spliced and matured and also can explain their different expression levels in different cells.
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Tollervey, James Robert. "Understanding misregulation of alternative splicing in the human TDP-43 proteinopathies." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609056.
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Herzel, Lydia. "Co-transcriptional splicing in two yeasts." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-179274.
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Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs.
Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered.
In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing.
I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes:
(1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs.
(2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data.
I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing.
The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS.
For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination.
Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.
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Öhrmalm, Christina. "Functional characterization of the cellular protein p32 : a protein regulating adenovirus transcription and splicing through targeting of phosphorylation /." Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6794.
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Carrillo, Catherine. "RNA splicing and editing of group II introns in flowering plant mitochondria." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/NQ57026.pdf.
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