xv, 106 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Myotonic Dystrophy type I (DM1) is caused by muscleblind protein sequestration to aberrantly expanded CUG repeats. When muscleblind is sequestered it can no longer fulfill its role as an alternative splicing regulator, leading to mis-splicing events in both humans and Drosophila . The muscleblind protein family's RNA binding specificity has been minimally characterized. Only one pre-mRNA target in humans, cardiac troponin T (cTNT), has a known MBNL1 binding site. In order to understand muscleblind's RNA binding specificity and identify a consensus binding motif, systematic evolution of ligands by exponential enrichment (SELEX) was performed on both the Drosophila muscleblind protein, Mbl, and the human ortholog, MBNL1. Drosophila has provided a useful model for studying the disease mechanism of DM1. Studies of Mbl's RNA binding specificity to CUG repeats concluded that replacing the U-U mismatches with different pyrimidine-pyrimidine mismatches was tolerated, but no other mutations were. To understand Mbl's RNA binding specificity, SELEX was performed. After 6 rounds, several sequences were identified that bound with high affinity, all containing the 5'-AGUCU-3' consensus motif. One sequence, SELEX RNA 20 was analyzed further. In addition to the guanosine in the consensus motif of SELEX RNA 20, two other guanosines were shown to be protected by Mbl in a footprinting assay, indicating that Mbl has a strong preference for binding guanosine. Also, two "tail" regions of SELEX RNA 20 were shown to be single stranded and required for binding by Mbl. These results indicate that Mbl is a highly specific RNA binding protein with preference for both single and double stranded guanosine-rich regions. A doped SELEX was performed on MBNL1's binding site from the cTNT pre-mRNA to determine which sequences and structural aspects were important for recognition by MBNL1. Pool 5 RNA sequences bound with high affinity, and the motif 5'-YGCUU-3' was selected. This motif was then used to identify new MBNL1 binding sites in pre-mRNAs regulated by MBNL1, SERCA1 and MBNL1. The identification of this motif and two new MBNL1 sites provide insight into MBNL1-mediated alternative splicing. This dissertation includes both my previously published co-authored material and my unpublished co-authored material. / Adviser: J. Andrew Berglund

xiv, 59 p. : ill. (some col.) / Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy (DM). In DM, the lack of properly localized MBNL1 leads to mis-splicing of many pre-mRNAs. The mechanism by which MBNL1 regulates it pre-mRNA targets is not well understood. In order to determine the mechanism by which MBNL1 regulates alternative splicing, a consensus RNA binding motif for Mbl (the <italic>Drosophila</italic> ortholog of MBNL1) and MBNL1 were determined using SELEX (Systematic Evolution of Ligands by Exponential Enrichment). These consensus motifs allowed for the identification of high affinity endogenous sites within pre-mRNAs that are regulated by MBNL1. <italic>In vitro</italic> binding studies showed that MBNL1 bound to RNAs that contained the consensus motif surrounded by pyrimidines. Some of these sites were identified upstream of exon 5 within the <italic>MBNL1</italic> pre-mRNA, and we have shown that MBNL1 auto-regulates the exclusion of exon 5 in HeLa cells. The region of the <italic>MBNL1</italic> gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3' end of intron 4 is non-canonical in that it contains an AAG 3' splice site and a predicted branchpoint that is 141 nucleotides from the 3' splice site. Using a mini-gene that includes exon 4, intron 4, exon 5, intron 5 and exon 6 of <italic>MBNL1</italic>, we show that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branchpoint confirms that branching occurs primarily at the predicted distant branchpoint. Structure probing and footprinting reveal that the highly conserved region between the branchpoint and the 3' splice site is primarily unstructured, and MBNL1 binds within this region of the pre-mRNA, which we have termed the MBNL1 response element. Deletion of the response element eliminates MBNL1 splicing regulation and leads to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing. This dissertation includes previously published co-authored material as well as my recent co-authored material that has been submitted for publication. / Committee in charge: Kenneth Prehoda, Chair; J. Andrew Berglund, Advisor; Victoria J. de Rose, Member; Alice Barkan, Member; Karen Guillemin, Outside Member

L'épissage alternatif est un processus biologique qui génère la diversité du protéome malgré le nombre limité de gène. Ce mécanisme régule à la fois les gènes de manières qualitatives (isoformes exprimées) mais aussi quantitatives (niveau d'expression). Avec le développement des technologies de séquençage à haut débit, il est maintenant possible d'étudier à large échelle les aspects quantitatif et qualitatif du transcriptome avec une même expérience (RNA-seq). Durant ma thèse, j'ai développé une nouvelle méthode d'analyse de l'épissage alternatif dans les données RNA-seq. J'ai également participé à la mise en place du pipeline global d'analyse de données RNA-seq (expression et épissage) qui a été utilisé pour analyser un grand nombre de jeux de données. Dans un second temps, nous avons comparé notre outil d'analyse de l'épissage, FaRLine, qui est basé sur l'alignement sur un génome de référence, à KisSplice, une méthode basée sur l'assemblage. Nous avons montré que ces méthodes trouvaient un grand nombre d'événements en communs (70%), mais qu'il existait des différences non négligeables dues à la méthodologie. Nous avons analysé et classifié ces événements en 4 grandes catégories. Les variants faiblement exprimés et les exons chevauchant des éléments répétés sont mieux annotés par les méthodes basées sur l'alignement. Alors que les méthodes basées sur l'assemblage trouvent des nouveaux variants (exons ou sites d'épissage non annotés) et prédisent de l'épissage alternatif dans les famille de gènes paralogues. Notre travail souligne les points qui nécessitent encore l'amélioration des méthodes bioinformatiques. Enfin, j'ai participé au développement de méthodes permettant d'aider les biologistes à évaluer l'impact fonctionnel de modification d'épissage, que ce soit au niveau de la protéine produite (annotation des domaines protéiques au niveau des exons), ou à un niveau plus global en intégrant les modifications d'épissage dans les voies de signalisation / Alternative splicing is the biological process that explain the large diversity of the proteome compared to the limited number of genes. This process allow a qualitative regulation (expressed isoforms) and a quantitative regulation (expression level). The growth of high-trhoughtput sequencing methods enabled the analysis of these two aspects (quantitative and qualitative regulation) with the same experiment (RNA-Seq). During my PhD, I developped a new tool to analyse alternative splicing from RNA-Seq data. I also participated in the automatisation of the complet pipeline of RNA-Seq analysis (expression and splicing). This pipeline has been used to analyse various datasets. Then, we compared our mapping-first tool, FaRLine, with an assembly-first method, KisSplice. We found that the predictions of the two pipelines overlapped (70\% of exon skipping events were common), but with noticeable differences. The mapping-first approach allowed to find more lowly expressed splicing variants, and was better in predicting exons overlapping repeated elements. The assembly-first approach allowed to find more novel variants, including novel unannotated exons and splice sites. It also predicted AS in families of paralog genes. Our work point out where the bioinformatic improvment are still needed. Finally, I participated in the developpement of bioinformatics methods to help biologists to evualuate the fonctionnal impact of splicing alteration : at the level of the protein product by annotating fonctionnal domain at the exon level or at a more global level, by integrating splicing modifications in signaling pathways

The post-natal regenerative capacity of skeletal muscle is attributed to myogenic satellite cells, which function as lineage-committed precursors to replace terminally differentiated muscle. The development and differentiation of the satellite cell lineage is regulated by Pax7 and the myogenic regulatory factors. While the expression of Pax7 is vital to the function of the satellite cell compartment, the paired domain alternative splicing events that regulate its DNA binding potential remain elusive. Interestingly, the generation of Pax7 splice variants differentially regulate Myf5 expression. We performed a global analysis of two Pax7 isoforms, which differ by a glycine-leucine dipeptide, to determine how paired domain splicing events modify the ability of Pax7 to regulate target genes. To this end, we observe that although the homeodomain is important for Pax7 binding, these isoform differences in the paired domain can regulate Pax7 targets during myogenesis. In addition to further examining the role of Pax7 during satellite cell proliferation and maintenance, it remains important to understand their downstream differentiation potential. Since activation of the canonical Wnt signalling pathway results in reduced regenerative efficiency in vivo, we undertook a global analysis of satellite cell derived myoblasts to examine their ability to respond to canonical Wnt signalling. We demonstrate that Wnt/β-Catenin signalling drives myogenic differentiation, via the myogenin-dependent control of follistatin expression, further fine-tuning the myogenic differentiation process. The effects of canonical Wnt signalling on myogenic differentiation complement our observations regarding Pax7 alternative splicing during myoblast proliferation and provide a greater comprehensive understanding of the molecular regulation of satellite cell development and differentiation during adult myogenesis.

The field of proteogenomics operates at the interface between proteomics and genomics, and has emerged during the past decade to exploit the vast quantities of high-throughput sequence data. A range of different proteogenomics approaches have been developed, which integrate mass spectrometry data with genome sequence data to provide empirical evidence for protein-coding genes. However, current methods may not be optimized as they do not fully consider the splicing complexity in eukaryotes and there is currently no best practice method. To address this, we investigate the level of proteomics support for Ensembl gene models in human, and a selection of model organisms. We find a disparity between the number of splice variants confirmed by extant data, and the number that can theoretically be confirmed using current proteomics technologies. We then go on to investigate EST-based proteogenomics methods, which enabled the discovery of novel peptide sequences in the chicken genome, which represent hitherto unannotated genes, amended gene models, polymorphisms, and genes missing from the genome assembly. Different approaches for searching mass spectrometry data against transcript sequences are explored, and we show that searching mass spectra against protein sequences predicted by the EORF and ESTScan2 translation tools results in the best sensitivity.

Bioinformática estrutural de proteínas modificadas por eventos de splicing alternativo / Structural Bioinformatics of Proteins modified by Alternative Splicing

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of pancreatic β-cells, mediated by autoreactive T cells.The resulting inflammatory response takes place in the context of a dialogue between invading immune cells and the targeted β-cells, and it is modulated by genetic susceptibility, acting on both immune and β-cells, and by inflammatory cytokines and chemokines. Stress pathways triggered within β-cells may potentiate autoimmunity, and T1D susceptibility genes shape β-cell responses to “danger signals”, innate immunity, and activation of apoptosis. However, the molecular mechanisms linking genetic variation, environmental triggers, and the signaling events promoting β-cell dysfunction and loss remain poorly clarified. Pre-mRNA splicing is a crucial mechanism for gene expression regulation, and more than 95% of the human multi-exonic primary transcripts undergo alternative splicing. Splicing dysregulation have been increasingly recognized to play a pivotal role in multiple pathologies, including autoimmune diseases. More than 15% of the mutations described in the Human Gene Mutation Database are predicted to affect splicing. Our group has shown that exposure to pro-inflammatory cytokines induces major changes on the β-cell transcriptome, affecting the splicing of genes that are key for β-cell function and survival. Importantly, our group identified that GLIS3, a susceptibility gene for both T1D and type 2 diabetes (T2D), modulates β-cell apoptosis via regulation of the splicing factor SRSF6, linking T1D genetic susceptibility and alternative splicing. The downregulation of GLIS3, either by germline mutations associated with monogenic forms of diabetes or risk single nucleotide polymorphisms, contribute to SRSF6 splicing factor downregulation. Splicing factors are the primary regulators of splicing and orchestrate functionally related transcripts into regulatory networks, therefore, oscillations of splicing factors’ expression levels have a major impact on splicing decisions. In the present study we aimed: 1. To evaluate the functional impact of SRSF6 downregulation in human pancreatic β-cells; 2. To identify the SRSF6-regulated splicing networks and to decode the SRSF6 cis-regulatory RNA binding elements.To fulfil these aims, human insulin-producing EndoC-βH1 cells were subjected to RNA sequencing (under control conditions or following SRSF6 knock down for 48h) to identify transcriptome-wide alternative splicing events regulated by SRSF6, and to individual-nucleotide resolution UV crosslinking and immunoprecipitation followed by high-throughput sequencing (iCLIP) to determine the SRSF6 mechanistic model of splicing regulation, its associated cis-regulatory elements and directly bound transcripts in human β-cells. We observed that SRSF6 depletion has a major impact on human pancreatic β-cell function and survival, leading to β-cell apoptosis and impaired insulin secretion. SRSF6 downregulation affects the splicing of transcripts involved in central pathways for β-cell function and survival, such as insulin secretion (e.g. INSR, SNAP25), apoptotic regulators (e.g. BCL2L11 (or BIM), BAX), and the mitogen-activated protein kinases (MAPKs) signaling pathway (e.g. MAPK8, MAPK9, MAP3K7). SRSF6 silencing potentiates the generation of constitutively active isoforms of pro-apoptotic inducers – BAX-β, and BIM-Small - leading to apoptosis activation, and also of different members of the MAPK signaling pathway contributing to the hyper-phosphorylation of the pathway, leading to activation of down-stream transcription factors and consequent β-cell apoptosis. These data indicate that specific splicing networks, regulated through diabetes susceptibility genes, control key pathways and processes involved in the function and survival of β-cells. The iCLIP analysis has shown that SRSF6 recognizes more than 100,000 of RNA binding sites in protein coding sequences, and it regulates splicing by preferentially binding into exons through a purine-rich consensus motif consisting of GAA triplets. The number of triplets in direct sequence correlates with increasing binding site strength. The SRSF6 binding position affects the splicing outcome, possibly resulting from the competition between alternative exons and their flanking constitutive exons for SRSF6 tethering. We identified SRSF6 binding sites on SRSF6-regulated cassette exons of several susceptibility genes for both T1D and T2D, and as a proof-of-concept, modulated the splicing of the LMO7 susceptibility gene using antisense oligonucleotides.In conclusion, our data suggest that SRSF6 is a master splicing regulator in pancreatic β-cells, downstream of the diabetes susceptibility gene GLIS3. SRSF6 silencing potentiates the splicing of constitutively active pro-apoptotic variants (BAX-β and BIM-Small), and exacerbates the MAPK signalling pathway. SRSF6 recognizes specific purine-rich RNA binding motifs, with important implications for the interpretation of sequence variants. This work unveiled a novel regulatory layer for β-cell demise and diabetes genetic susceptibility, namely through splicing mis-regulation. These observations raise the possibility that splicing networks regulated by candidate genes for diabetes contribute to β-cell dysfunction and death in diabetes. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Pre-mRNA splicing is the most critical process in gene expression regulation across eukaryotic species. This reaction is carried out by the spliceosome, a large, dynamic, and well-organized ribonucleoprotein complex. The spliceosome is composed of five major small nuclear RNAs and an excessive number of associated protein factors. Many protein splicing factors bind and release during splicing to assist the assembly and the modulation of many RNA structures and proteins within the spliceosome. CWC25 is a splicing protein factor that functions in modulating the conformational structure of the spliceosome at the first transesterification reaction. CWC25 binds with its N-terminus to the major groove of the catalytic spliceosome triggering the spliceosome activity. Here, we employed CRISPR/Cas9 genome engineering system for targeted mutagenesis to generate CWC25 functional knock-out mutants to understand its molecular function, contribution to splicing regulation and implication in fine-tuning responses to abiotic stress in rice. Our genotyping analysis of the OsCWC25 locus revealed the presence of two mono-allelic and 18 bi-allelic mutant lines. Phenotypic analysis of these mutants, including germination and root inhibition assays, showed that the cwc25 mutants are oversensitive to abiotic stresses such as ABA and salinity. Our data demonstrate that CWC25 plays an important role in regulating plant responses to abiotic stresses.

At the post-transcriptional level, the splicing of the pre-mRNA plays a vital role in cell fate determination and respond to biotic and abiotic stresses. Through alternative splicing, mRNAs variants can be produced from a single gene. SF3B is a heptameric protein complex that is essential for pre-mRNA splicing. It contains seven subunits: SF3b155, SF3b130, SF3b145, SF3b49, SF3b14b, P14/SF3b14a and SF3b10 and they play an important role in BS (branch point sequence) recognition. P14/SF3b14a interacts with the branch point Adenosine (BPA), directing the binding of U2 complex. Several studies performed on the mutations of SF3b complex as it is associated with many diseases. Further studies are needed to deeply analyze the molecular function of P14/SF3b14a in plant growth and development. CRISPR/Cas9 system employed in gene editing among eukaryotes. The capability of the system is not only limited to the scope of bioengineering but also for functional studies of genes. CRISPR/Cas9 system assists in revealing the function of genes and the genetic networks through establishing a functional knockout and can help in understanding the molecular basis behind these processes. Here, we report the successful targeted mutagenesis of SF3b14a/p14 gene in Oryza sativa and the recovery of homozygous and heterozygous mutants. Phenotypic analyses have shown that SF3b14a/p14 is hypersensitive to abiotic stresses compared to the wild type plants. Further physiological and molecular studies are needed to reveal the role of p14 during plant growth and development, and responses to abiotic stresses.

Plants acclimate to the changing environmental conditions by adjusting their molecular responses at different molecular levels including genome, epigenome, transcriptome, metabolome, and proteome levels to ensure survival. Plants adapt to abiotic stresses by establishing a ‘stress memory’ of previous exposures to mild stresses. Stress memory helps plants to develop tolerance and survive recurring exposures to the stress conditions. This memory establishes a new cellular state that differs from the state of unexposed naïve plants. This process is known as priming. Priming and the stress memory give the plants the possibility to acclimate to different biotic and abiotic stress conditions. The acquisition and maintenance of the stress-memory are two separate processes and crucial for successful tolerance to subsequent stress conditions. Priming promises to improve plant performance under severe stress conditions and enhance food production. Therefore, understanding the molecular basis of heat stress priming and stress-induced memory is of vital importance to plant biology. In this thesis, I investigated the role of transcriptional, post-transcriptional and metabolomic regulation controlling plant responses to heat stress, one of the major abiotic stresses affecting agriculture. I designed and established a heat stress priming strategy which reveals that heat stress-induced priming leads to the establishment of heat stress memory that permits plants to survive lethal temperatures. In this thesis, I analyzed the genome-wide differential gene expression, the alternative splicing patterns and regulation, and the reprogramming of the metabolic homeostasis that reprogram the establishment of the heat stress priming and stress-memory. I identified a set of candidate genes and metabolites playing key roles in the establishment of heat stress-induced memory. Intriguingly, it was possible also to establish a link between alternative splicing patterns and heat stress-induced memory. Subsequently, the knowledge of heat stress priming in Arabidopsis was translated into tomato crop plants, to improve their heat stress tolerance. This work enhances our understanding of the molecular basis of heat stress priming, and the establishment of heat stress memory, at transcriptional, post-transcriptional, and metabolomic levels. These findings can be translated into crop species to improve their survival under recurring heat stress conditions to improve world agriculture and food security.