Magister Scientiae - MSc (Biochemistry) / Alternative RNA splicing is one of the leading mechanisms contributing towards transcript and protein diversity. Several alternative splicing surveys have confirmed the frequent occurrence of exon skipping in human genes. However, the occurrence of exon skipping in mouse genes has not yet been extensively examined. Recent improvements in mouse genome sequencing have permitted the current study to explore the occurrence of exon skipping in mouse genes orthologous to human genes on chromosome 22. A low number (5/72 multi-exon genes) of mouse exon-skipped genes were captured through alignments of mouse ESTs to mouse genomic contigs. Exon-skipping events in two mouse exon-skipped genes (GNB1L, SMARCB1) appear to affect biological processes such as electron and protein transport. All mouse, skipped exons were observed to have ubiquitous tissue expression. Comparison of our mouse exon-skipping events to previously detected human exon-skipping events on chromosome 22 by Hide et al.2001, has revealed that mouse and human exon-skipping events were never observed together within an orthologous gene-pair. Although the transcript identity between mouse and human orthologous transcripts were high (greater than 80% sequence identity), the exon order in these gene-pairs may be different between mouse and human orthologous genes. Main factors contributing towards the low detection of mouse exon-skipping events include the lack of mouse transcripts matching to mouse genomic sequences and the under-prediction of mouse exons. These factors resulted in a large number (112/269) of mouse transcripts lacking matches to mouse genomic contigs and nearly half (12/25) of the mouse multi-exon genes, which have matching Ensembl transcript identifiers, have under-predicted exons. The low frequency of mouse exon skipping on chromosome 22 cannot be extrapolated to represent a genome-wide estimate due to the small number of observed mouse exon-skipping events. However, when compared to a higher estimate (52/347) of exon skipping in human genes for chromosome 22 produced under similar conditions by Hide et al.2001, it is possible that our mouse exon-skipping frequency may be lower than the human frequency. Our hypothesis contradicts with a previous study by Brett et al.2002, in which the authors claim that mouse and human alternative splicing is comparable. Our conclusion that the mouse exon-skipping frequency may be lower than the human estimate remains to be tested with a larger mouse multi-exon gene set. However, the mouse exon-skipping frequency may represent the highest estimate that can be obtained given that the current number (87) of mouse genes orthologous to chromosome 22 in Ensembl (v1 30th Jan. 2002) does not deviate significantly from our total number (72) of mouse multi-exon genes. The quality of the current mouse genomic data is higher than the one utilized in this study. The capture of mouse exon-skipping events may increase as the quality and quantity of mouse genomic and transcript sequences improves. / South Africa

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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.

Members of the Mef2 transcription factor family are extensively studied within the muscle field for their ability to cooperate with the myogenic regulatory factors MyoD and myogenin during muscle differentiation. Although it is known that Mef2 pre-mRNAs undergo alternative splicing, the different splice forms have not been functionally annotated. In this thesis, my studies aimed to characterize three Mef2D splice forms: MEF2Dα'β, MEF2Dαβ, MEF2Dαø. Our results show that MEF2D splice forms can be differentially phosphorylated by p38 MAPK and PKA in vitro. Gene expression analysis using cell lines over-expressing each Mef2D splice form suggests that they can differentially activate desmin, myosin heavy chain and myogenin expression. Mass spectrometry analyses from our pull-down assays reveal known and novel MEF2D binding partners. Our work suggests that Mef2D splice forms have overlapping but distinct roles and provides new insight into the importance of Mef2D alternative splicing during skeletal myogenesis.

A subset of mitochondrial group II introns of flowering plants has, over evolutionary time, lost characteristic features and employs unconventional splicing pathways. Given the potential impact of cold treatment on RNA folding, as well as on enzymatic activity and import of nuclear-encoded splicing machinery, I have examined the physical excised forms of aberrant introns from wheat embryos subjected to 4oC. My findings suggest a shift in biochemistry with cold treatment to novel splicing pathways that generate heterogeneous in vivo circularized forms for nad1 intron 2, nad2 intron 1 and the cox2 intron, in contrast to predominantly linear excised intron forms at room temperature. Interestingly, the highly degenerate nad1 intron 1, which due to DNA rearrangement has been broken into two halves that interact for splicing in trans, is excised exclusively by first-step hydrolysis at room temperature and under cold treatment. In this case, splicing culminates in two distinct linear half introns that appears correlated with an unusual 5’ terminal insert. This represents the first in vivo demonstration of hydrolytic trans-splicing. Based on northern analysis, cold treatment was further associated with reduced splicing efficiency for all introns surveyed. Moreover, study of precursor transcripts of the nad1a-intron 1a locus suggests the efficiency of end-maturation, including processing of the cotranscribed tRNA-Pro gene, is also reduced in the cold. My findings demonstrate a temperature-sensitivity of transcript maturation, particularly for RNA splicing, providing new insight into the impact of cold growth conditions on plant mitochondrial gene expression.

The U5 snRNP is a major component of the yeast spliceosome, being part of the U4/U6.U5 tri-snRNP, the precatalytic spliceosome and the catalytically activated spliceosome. The U5 snRNP includes, at its heart, the U5 snRNA which contains the invariant Loop 1 that functions in tethering and aligning exons during splicing. The major protein components of the U5 snRNP are the highly conserved Prp8p, the GTPase Snu114p and the helicase Brr2p. These proteins and the U5 snRNA are integral in forming the active site of the spliceosome and regulating the dynamic changes of the spliceosome. The first part of this study aimed to express and purify specific domains of Snu114p to define the structure and function of Snu114p. The N-terminal region of Snu114p was successfully expressed and purified from bacteria. Addition of the Snu114p N-terminal fragment to in vitro splicing assays resulted in a first step splicing defect, indicating a role for the N-terminus in pre-mRNA splicing. NMR studies revealed that the N-terminus of Snu114p exists as an unstructured protein domain. Mutagenesis indicated that the N-terminus of Snu114p is tolerant to mutation. A novel genetic interaction between amino acids in the N-terminus of Snu114p and the 3’ side of the U5 snRNA IL1 was identified. It is proposed here that the N-terminus of Snu114p functions to stabilise interactions of Snu114p with other proteins or snRNAs, possibly the U5 snRNA. Alternatively, the N-terminus of Snu114p may form intramolecular interactions with other regions of Snu114p to regulate Snu114p function in pre-mRNA splicing.Prp8p, Snu114p and Brr2p are known to form a stable complex but their interactions with the specific domains of the U5 snRNA are not known. The second part of this study aimed to investigate the association of Brr2p, Snu114p and Prp8p with the U5 snRNA. Mutants of the U5 snRNA were constructed in the conserved Loop 1 and the Internal Loop 1 (IL1). The influences of the U5 snRNA mutations on interactions of Prp8p, Snu114p or Brr2p with the snRNA were investigated. It was revealed that Loop 1 and both sides of IL1 of the U5 snRNA are important in association of Brr2p, Snu114p and Prp8p. Mutations in the 3’ side of IL1 drastically reduce association of Brr2p, Snu114p and Prp8p with the U5 snRNA, highlighting this region as a potential ‘protein docking’ site within the U5 snRNP. Differences seen in the associations of Brr2p, Snu114p and Prp8p with U5 snRNA mutations demonstrate that although there are intimate interactions between Brr2p, Snu114p and Prp8p, they do not associate with the U5 snRNA as a tri-protein complex. Genetic screening of BRR2 and U5 snRNA mutants reveals an interaction between the N-terminal half of Brr2p and the 3’ side of U5 snRNA IL1. This supports the proposed ‘protein docking’ site at the 3’ side of the U5 snRNA IL1.Data presented in this study increases our understanding of the regions in the U5 snRNA required for association of the essential U5 snRNP proteins, Brr2p, Snu114p and Prp8p, and goes some way to elucidating the organisation of essential proteins within the U5 snRNP.

Splicing is the process where pre-mRNA is converted to mRNA via two transesterification reactions. With this process unwanted sequences of nucleic acids, known as introns, are removed allowing only the coding nucleic acid sequences, exons, to remain. This process is catalysed by a dynamically assembled, highly complex macromolecular machine called the spliceosome, which is made up of five small nuclear ribonucleoproteins (snRNPs). To date, the spliceosome has defied conventional methods for conclusive characterisation, resulting in it being relatively poorly understood, although advances have been made.1, 2 Apart from being of interest due to the fact that splicing is an essential life process, it is also of interest medically. Disruption to the splicing process can produce incorrectly formed mRNA, which plays a part in many diseases.3 Small molecule inhibitors which bind to, and inhibit, the functions of individual proteins would “stall” the spliceosome,4 circumventing its dynamic nature. These inhibitors could also form the basis of new drugs, treating diseases which incorrectly formed mRNA can cause. Previously reported small molecule inhibitors have inhibited splicing at the early stages of spliceosome assembly.5-7 However, our target protein snu1148 belongs to the U5 snRNP, which is involved later on in the splicing cycle. Inhibition of Snu114 should, therefore, lead to accumulation of spliceosome complexes produced at later stages of the cycle. Homology studies of Snu114 indicated a strong correlation of amino acid sequences with ribosomal growth factors EF-2 and EF-G. This study allowed us to target Snu114 using known EF-2 and EF-G inhibitors, sordarin and fusidic acid, which were tested and found to have significant splicing inhibition activity. A series of derivatives of these parent compounds were then attempted in an effort to improve splicing inhibition activity and to analyse the structure-activity relationship of fusidic acid and sordarin as splicing inhibitors. The biosynthesis of sordarin proved to be difficult and only a few derivatives were synthesised, however an improvement was made to splicing inhibition activity by forming sordaricin 32. Various fusidic acid derivatives were successfully synthesised, leading to an analysis of the structure-activity relationship of fusidic acid as a splicing inhibitor. Most fusidic acid derivatives produced a lower splicing inhibition activity than fusidic acid. However, fusidic acid derivative 229 had an equivalent inhibition activity to that found for fusidic acid. This result leads us to believe that the C-3 hydroxyl moiety of fusidic acid would be an ideal area for modification in future studies.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-11T08:45:17Z No. of bitstreams: 1 texto completo.pdf: 2070709 bytes, checksum: 8aca4e1bccb3e3a726679695fe3ab253 (MD5) / Made available in DSpace on 2015-11-11T08:45:17Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2070709 bytes, checksum: 8aca4e1bccb3e3a726679695fe3ab253 (MD5) Previous issue date: 2014-08-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Serine-Arginine Protein Kinase 2 - SRPK2 é uma cinase reguladora de fatores de splicing não-snRNPs (proteínas SR), e de fatores snRNPs (U4⁄U6-U5 tri-snRNP). SRPK2 regula as proteínas SR fosforilando-as em seus resíduos de serina e recrutando- as para a formação do spliceossomo, enquanto atua na formação do complexo U4⁄U6- U5 tri-snRNP, envolvido na seleção do sítio de splicing γ’. As interações com fatores snRNP e não-snRNP são conhecidas e bem estabelecidas funcionalmente, no entanto, pouco se sabe sobre proteínas que regulam o funcionamento da SRPK2, e sobre demais alvos de fosforilação. As proteínas SRPKs possuem seu domínio cinase divido em dois por uma região espaçadora de grande flexibilidade estrutural, passível de interação. Portanto, possível responsável pela seleção de substratos e/ou alvo de regulação. Nosso objetivo com este trabalho foi encontrar novas interações proteicas de SRPK2 através de sua região espaçadora. A técnica escolhida foi o duplo-híbrido em leveduras, no qual estas foram cotransformadas com o plasmídeo pBTM116K/S-SRPK2 trp1 e com uma biblioteca de cDNAs de leucócitos humanos em pACT2 leu2. O experimento foi processado em meio restritivo SD–TRP –LEU –HIS, acrescido de 30mM de 3AT e mantido em estufa 30°C por 120 horas. Ao final deste período, foi verificado o crescimento de 119 colônias de leveduras, submetidas a diferentes testes de ativação dos genes repórteres his3 e lacZ. Os resultados destes testes nos levaram a seleção de 42 clones, que tiveram seus DNAs plasmidiais extraídos, sequenciados e analisados pela ferramenta Blast do NCBI. As proteínas codificadas por estes genes estão funcionalmente relacionadas a processos celulares como biogênese ribossomal, migração, diferenciação, angiogênese, proliferação, sobrevivência e ciclo celular. Estes resultados sugerem novos mecanismos moleculares para a atuação da SRPK2 no processo tumorigênico. / Serine-Arginine Protein Kinase 2 - SRPK2 is a regulator kinase of non-snRNP splicing factors (SR proteins) and of snRNPs factors (U4⁄U6-U5 tri-snRNP). SRPK2 regulates SR proteins phosphorylating them on their serine and recruiting them for the formation of the spliceosome, while acts on the tri-snRNP U4/U6-U5 complex assembly, involved in the splicing γ’ site selection. The interactions with snRNP and non-snRNP factors are known and well-established functionally, however, little is known about the proteins that regulate the functioning of SRPK2, and about other targets of phosphorylation. The SRPKs proteins have their kinase domain divided into two by a spacer region of great structural flexibility, believable of interaction. Therefore, possible responsible for selection of substrates and/or aim of regulation. Our aim with this work was to find new protein interactions of SRPK2 by its space region. The technique chose was two-hybrid yeast, in which yeasts were cotransfected with pBTM116K/S-SRPK2 trp1 plasmid and a human leukocyte cDNA library in pACT2 leu2. The experiment was processed in restrictive medium SD –TRP –LEU –HIS, plus 30 mM of 3AT and keep in a incubator 30°C for 120 hours. At the end of this period, it was found the growth of 119 yeast colonies, subjected a different tests of activation of reporter genes his3 and lacZ. The results of these tests led to the selection of 42 clones, which had its plasmid DNA extracted, sequenced and analyzed by the NCBI Blast tool. The proteins encoded by these genes are functionally related in cellular processes as ribosome biogenesis, migration, differentiation, angiogenesis, proliferation, survival and cell cycle. These results suggest new molecular mechanisms for the performance of SRPK2 in tumorigenic process. / Sem lattes

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LSD1 is the first described histone demethylase which demethylates H3K4me1/2 (Shi et el., 2004), thus, causing transcriptional repression. Alternatively, LSD1 was demonstrated to have H3K9me1/2 demethylase activity when bound by androgen receptor, hence, causing transcriptional activation (Schule et al., 2005). LSD1 is commonly recruited by the so called CoREST core complex including: RCOR1, HDAC1 and HDAC2 among others and therefore is coupled with histone deacetylation and transcriptional repression (Foster et al., 2010). It is an important regulator of pluripotency in early development and it occupies, along with pluripotency factors NANOG and OCT4, the promoters of major lineage determining genes that are poised for activation in the pluripotent state, (Adamo et al., 2011). There are four described isoforms for LSD1: LSD1, LSD1-E2a, LSD1-8a and LSD1-E2a/E8a (Zibetti et al., 2010). While the Cterminus of LSD1 is extensively studied and the function of the isoforms LSD1-E8a and LSD1-E8aE2a is described, there is scarce knowledge on LSD1 N-terminus unstructured region and the SWIRM domain. In this project I examined the role of the differently spliced exon 2a on the function of the SWIRM domain through generation of eight constructs coding for the N-terminal portion of LSD1 SV1 and SV2 fused with a C- or N-terminus FLAG tag. I then performed an immunoprecipitation experiment followed by mass spectrometry and proteomics analysis that led to the identification of previously unknown binding partners to the LSD1 SWIRM domain: NONO and IGF2B3.