Splicing is the process when introns gets removed and exons are spliced together. This is an important step to form a clean mRNA with no unnecessary sequences that could interrupt protein synthesis. There are different types of splicing and some of them need a complex called spliceosome. The spliceosome requires ATP, small nuclear RNAs and splicing factors. The spliceosome and the process splicing can be regulated by epigenetics, and one epigenetic mechanism is histone modification. There are four types of histone modifications; methylation, phosphorylation, ubiquitination and acetylation. They regulate splicing to different extents by altering the chromatin structure, affect the assembly of the spliceosome and regulate the attraction of splicing factors. This review will investigate if histone modifications affect splicing and to what extent. Suggestions for further research regarding the relationship between splicing and histone modifications will also be provided. The review is based on 30 articles and two books and the search was conducted between 30th of March 2020 and 13th of April 2020. Ubiquitination and phosphorylation have a minor effect on splicing meanwhile methylation and acetylation affect splicing in great extent.

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Yes / It has long been known that Tissue Factor (TF) plays a role in blood coagulation and has a direct thrombotic action that is closely related to cardiovascular risk, but it is becoming increasingly clear that it has a much wider range of biological functions that range from inflammation to immunity. It is also involved in maintaining heart haemostasis and structure, and the observation that it is down-regulated in the myocardium of patients with dilated cardiomyopathy suggests that it influences cell-to-cell contact stability and contractility, and thus contributes to cardiac dysfunction. However, the molecular mechanisms underlying these coagulation-independent functions have not yet been fully elucidated. In order to analyse the influence of TF on the cardiomyocitic proteome, we used functional biochemical approaches incorporating label-free quantitative proteomics and gene silencing, and found that this provided a powerful means of identifying a new role for TF in regulating splicing machinery together with the expression of several proteins of the spliceosome, and mRNA metabolism with a considerable impact on cell viability.

Most of the protein coding genes of higher eukaryotes contain introns which have to be removed from primary transcripts to make mRNA which can be used as a template for protein synthesis. This crucial step in the pre-mRNA processing is carried out by the spliceosome, a complex ribonucleoprotein machine formed from small ribonucleoprotein particles (snRNPs). snRNPs biogenesis is a complex process composed of several steps which take place in both the cytoplasm and the nucleus. Spliceosome assembly is highly dynamic and tightly regulated and pre-mRNA splicing depends not only on the sequence of the pre-mRNA itself but also on the nuclear context, such as the chromatin modifications. How do cells regulate where and when the spliceosome would be assembled? What determines which introns will be spliced? These are fundamental, yet unanswered, biological questions. In this work we analyzed the formation of splicing machinery in the context of the cell nucleus from several different points of view. First, we investigated the unexpected connection between splicing factor U1-70K and the survival of motor neurons (SMN) complex which is a major player in the snRNP biogenesis pathway. We revealed that U1-70K interacts with the SMN complex and that this interaction is crucial for the stability of nuclear gems, small...

O SL trans-splicing (do inglês, spliced leader trans-splicing) catalisado pelo spliceossomo em Trypanosoma brucei é responsável pelo processamento dos pré-mRNAs policistrônicos em mRNAs maduros. Esta maquinaria é associada a partir de pequenas partículas ribonucleoproteicas nucleares (snRNPs) U1, U2, U4/U6 e U5 constituídas de pequenos RNAs nucleares (snRNAs), um complexo canônico de sete proteínas Sm (SmB, SmD3, SmD1, SmD2, SmE, SmF e SmG) e fatores proteicos específicos. O núcleo de proteínas Sm de T. brucei apresenta variações com funções desconhecidas, como a substituição do heterodímero SmD3/SmB por Sm16,5K/Sm15K na snRNP U2, e de SmD3 por SSm4 na snRNP U4. Na primeira parte deste trabalho, investigou-se a interação destes diferentes complexos Sm recombinantes com os snRNAs U2, U4 e U5 obtidos por transcrição in vitro. Todos os complexos apresentaram alta afinidade pelo snRNA cognato. Observou-se, ainda, que apenas o núcleo Sm que contém Sm16,5K/Sm15K associado ao snRNA U2 interage com alta afinidade com U2A/U2B. Adicionalmente, foi obtida a estrutura cristalográfica de U2A/U2B de T. brucei, que revela uma organização similar àquela já descrita para ortólogas de Homo sapiens. Entretanto, há um desvio de pelo menos 6 Å no ponto médio da alça carregada positivamente no domínio RRM de U2B para a acomodação do snRNA U2. Além disso, observou-se uma longa hélice-α adicional na extremidade C-terminal de U2A. A análise dos três núcleos Sm de T. brucei a partir da combinação de modelagem molecular e espalhamento de raios-X a baixo ângulo revela estruturas de barril-β altamente torcido com interior carregado positivamente para interação com snRNAs. A principal diferença entre as estruturas encontra-se nas extremidades C- e N-terminal dos variantes de proteínas Sm, possivelmente para interação com U2A no braço 1 do spliceossomo, no caso de Sm15K/Sm16,K, e com U5-220K e U5-200K no corpo do spliceossomo, no caso de SSm4. Na segunda parte deste trabalho, a expressão homóloga da proteína U5-200K de T. brucei completa e do produto truncado no seu cassete helicase/ATPase/Sec63 N-terminal levou à copurificação de um subcomplexo de snRNP U5 composto por U5-220K, U5-116K, U5-40K e U5-Cwc21, sendo que a proteína recombinante completa ainda copurificou as proteínas Sm. Experimentos de imunolocalização mostraram que a proteína U5-200K truncada não é direcionada ao núcleo, como é o caso da proteína completa. As células que expressam a proteína truncada apresentaram um defeito de crescimento significativo, e os processamentos de pré-mRNA por cis- e SL trans-splicing foram ligeiramente afetados, já que a proteína truncada não entra no núcleo, onde deveria exercer sua atividade. Os resultados apresentados indicam a formação de um subcomplexo de snRNP U5 ainda no citoplasma, sendo que as proteínas Sm devem ser um sinal para o seu transporte nuclear mediado por importina-β. Em leveduras, a proteína Aar2 substitui U5-200K no citoplasma, regulando assim a biogênese de snRNP U5, porém esta proteína não foi identificada em T. brucei. Os resultados apresentados neste trabalho contribuem como o primeiro estudo estrutural de proteínas spliceossomais de um parasita do homem e também com novas informações sobre a biogênese das partículas ribonucleoproteicas U2 e U5 de T. brucei. / The spliced-leader (SL) trans-splicing catalyzed by the spliceosome in Trypanosoma brucei is responsible for processing polycistronic pre-mRNAs into mature mRNAs. The spliceosome machinery is assembled by small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 that are composed by small nuclear RNAs (snRNAs), a canonical complex of Sm proteins (SmB, SmD3, SmD1, SmD2, SmE, SmF, SmG) and specific factors. The Sm core peculiarly varies in T. brucei, where SmD3/SmB are replaced by Sm16.5K/Sm15K in U2 snRNP and SmD3 is substituted by SSm4 in U4 snRNP. In the first part of this thesis, we investigated the interaction of the different recombinant Sm cores with U2, U4 and U5 snRNAs obtained by in vitro transcription. All the protein complexes bind the cognate snRNA with high affinity. Only the Sm core that contains Sm16.5K/Sm15K associated with U2 snRNA interacts with the recombinant U2A/U2B subcomplex. Additionally, the crystallographic structure of T. brucei U2A/U2B was obtained, showing an overall organization similar to the one observed in the human counterpart. However, we observed a 6 Å deviation in the medium point of a positively charged turn in the RRM motif of U2B to accommodate U2 snRNA. Besides, a long α -helix was observed in the C-terminal region of U2A. Structural analysis of Sm core variations in T. brucei was proceeded using molecular modelling techniques associated with small angle X-ray scattering. The quaternary structure models show seven Sm proteins as β-barrels with positively charged interior for cognate snRNA interaction. The main difference among these Sm core structures resides in the C- and N-terminal regions of the variant proteins, probably enabling the interaction of Sm15K/Sm16,5K with U2A in the spliceosomes arm 1, and the association of SSm4 with U5-220K and U5-200K in the spliceosomes body. In the second part of this thesis, homologous expression of full-length and N-terminally truncated U5-200K from T. brucei led to the copurification of a U5 snRNP subcomplex containing U5-220K, U5-116K, U5-40K and U5-Cwc21. The full-length U5-200K construct also copurified Sm proteins. Immunolocalization experiments showed that the truncated U5-200K protein is not directed to the nucleus as is the case for the full-length protein. Cells that expressed the truncated protein showed a significant growth defect and the pre-mRNA processing by cis- and SL trans-splicing was negatively affected since the truncated protein did not enter the nucleus where it should be active. The results suggest that a subcomplex of U5 snRNP begins to be assembled in the cytoplasm and the Sm proteins may be the signal for the nuclear transport mediated by β-importin. In yeast, Aar2 replaces U5-200K in the cytoplasm in another regulation step. However, Aar2 has not been identified in T. brucei. The results presented here contribute with the first structural study of spliceosomal proteins of a human parasite and give new insights into the biogenesis of U2 and U5 snRNPs in T. brucei.

Pre-mRNA (precursor messenger RNA) splicing is a fundamental process in eukaryotic gene expression. In order to catalyse the excision of the intervening intronic sequence between two exons, the spliceosome is assembled stepwise on the pre-mRNA substrate. This ribonucleoprotein machine is extremely dynamic: both its activation and the progression through the catalytic stages require extensive compositional and structural remodelling. The first part of this thesis aims at understanding how the spliceosome is activated after assembly. When this work was started, the GTPase Snu114 was thought to activate the helicase Brr2 to unwind the U4/U6 snRNA duplex, which ultimately leads to the formation of the spliceosome active site. To explore the role of Snu114, a complex built from Snu114 and a part of Prp8 was expressed and analysed in its natural context, bound to U5 snRNA. However, before I was able to obtain highly diffracting crystals, the structure of Snu114 was determined in the context of a larger spliceosomal complex by electron cryo-microscopy by competitors. Regardless, the role of Snu114 in spliceosome activation remains elusive. In a short section of this thesis, genetic and biochemical analysis suggest Snu114 to be a pseudo-GTPase, precluding a role for Snu114-catalyzed GTP hydrolysis in activation. The second and larger part of the thesis describes the development of a novel, biochemical method to analyse spliceosome remodelling events that are caused by the eight spliceosomal helicases. Purified spliceosomes assembled on a defined RNA substrate are analysed by UV crosslinking and next-generation sequencing, which allows for the determination of the RNA helicase binding profile at nucleotide resolution. In vitro spliceosome iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) was initially developed targeting the helicase Prp16 bound to spliceosomal complex C. The obtained binding profile shows that Prp16 contacts the intron, about 15 nucleotides downstream of the branch in the intron-lariat intermediate. Our finding supports the model of Prp16 acting at a distance to remodel the RNA and protein interactions in the catalytic core and thereby it promotes the transition towards a conformation of the spliceosome competent for second step catalysis. Control experiments, which locate SmB protein binding to known Sm sites in the spliceosomal snRNAs, validated the method. Preliminary results show that in vitro spliceosome iCLIP can be adapted to analyse additional spliceosomal helicases such as Prp22. Finally, I performed initial experiments that give promising directions towards time-resolved translocation profiles of helicases Brr2 and Prp16.

Nuclear pre-mRNA splicing proceeds via two mechanistically conserved consecutive trans-esterification reactions catalyzed by the spliceosome. The ordered coalescence of spliceosomal snRNPs and splicing factors on the pre-mRNA, coupled with essential spliceosomal rearrangements poise the splice-sites in proximity for the two catalytic reactions, ensuring intron removal and exon ligation to yield functional mRNA (reviewed in Will and Lührmann, 2006). Scope of the study The S. cerevisiae splicing factors Prp18 and Slu7 and their human homologs function during second catalytic reaction. In S. cerevisiae, Slu7 is essential, whereas Prp18 is dispensable at temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Frank et al., 1992; Horowitz and Abelson, 1993b; reviewed in Umen and Guthrie, 1995). Slu7 acts in concert with Prp18 and their direct interaction is required for their stable spliceosomal association (Zhang and Schwer, 1997; James et al., 2002). In vitro studies indicate that both the factors are dispensable for splicing of introns with short distances between branch nucleotide to 3’ splice-site (Brys and Schwer, 1996; Zhang and Schwer, 1997). Furthermore, mutational analyses of Slu7 and Prp18 have defined their functional domains/motifs (Frank and Guthrie, 1992; Bacíková and Horowitz, 2002; James et al., 2002). In this study, we have examined functions for the predicted homologs of Slu7 and Prp18 in fission yeast; an evolutionarily divergent organism where splicing mechanisms are not well understood and whose genome harbors genes with predominantly multiple introns with degenerate splice-junction sequences. Towards this goal, a combinatorial approach employing genetic and biochemical methods was undertaken to understand splicing functions and interactions of SpSlu7 and SpPrp18. Our mutational analysis of these protein factors provided an overview of the domains/motifs critical for their in vivo functions. Lastly our analysis of components of the budding yeast Cef1p-associated complex show novel interactions and splicing functions for two uncharacterized, yet evolutionarily conserved proteins. Conserved fission yeast splicing proteins SpSlu7 and SpPrp18 are essential for pre-mRNA splicing but have altered spliceosomal associations and functions Analyzing conserved splicing factors in evolutionarily divergent organisms is an important means to gain deeper functional insights on splicing mechanisms in genomes with varied gene architecture. We initiated our analysis of the ‘predicted’ S. pombe second-step splicing factors: spprp18+ and spslu7+, by genetically depleting these factors. We find spprp18+ is essential for viability, unlike budding yeast PRP18; while SLU7 is essential in both yeasts. The complete essentiality of both these fission yeast factors, prompted us to create conditional-lethal thiamine repressible ‘switch-off’ strains to probe their splicing functions. Through semi-quantitative RT-PCR and northern blot analysis we demonstrate splicing defects for tfIId+ pre-mRNAs upon metabolic depletion of spprp18+ or spslu7+, thus linking their essentiality to a role in pre-mRNA splicing. Further we examined whether their requirement as splicing factors is governed by specific intronic features. We find both factors are required in vivo for removal of several introns. However, for the introns tested, their functions are not strictly correlated with intron length, number, position or the branch-nucleotide to 3’ splice-site distance. The latter features dictate the need for their S. cerevisiae homologs. Strikingly the lack of either one of these essential proteins, arrests splicing before the first catalytic step; implicating possible functions early in spliceosome assembly even before any catalytic event, as opposed to budding yeast Slu7 and Prp18, which are second-step factors assembling late onto the spliceosome after the first splicing reaction. Given the different splicing arrest point, on depletion of SpSlu7 and SpPrp18, we investigated through yeast two-hybrid and co-immunoprecipitation assays whether the direct interaction between these proteins is conserved. We find despite being nuclear localized these proteins do not interact in either of the assays employed. A structural basis for the lack of interaction was provided by our homology modeling of SpPrp18, that was based on the crystal structure of S. cerevisiae Prp1879 (Jiang et al., 2000). Together these data raise the possibility of contextual functions and interactions for these conserved proteins that varies with changes in gene architecture. This likelihood is strengthened by our reciprocal genetic complementation tests; wherein we find that SpSlu7 and SpPrp18 cannot complement the corresponding S. cerevisiae null alleles and vice versa. Additionally, the human homologs, hSlu7 and hPrp18 also failed to rescue null alleles for spslu7+ and spprp18+. To understand the likely point of coalescence of SpSlu7 and SpPrp18 on assembling spliceosomes, we probed their snRNP associations through co-immunoprecipitation analysis. Our data revealed interaction of SpSlu7 with the U2, U5 and U6 snRNPs at moderate salt concentrations with the interaction with U5 snRNP being retained at higher salt conditions. SpPrp18, on the other hand, showed only a very weak association with U5 snRNP. Our analysis thus indicates that the assembly and step of action for “predicted” late-acting splicing factors in fission yeast differs from that in budding yeast, implicating novel interactions and functions for these fission yeast splicing factors. Mutational analysis of fission yeast SpPrp18 and SpSlu7 identifies functional domains To examine the protein domains/motifs critical for the functions of SpPrp18 and SpSlu7, we have performed a mutational study. This analysis was important after our findings that these factors are early acting and do not interact. The data gathered would shed light on the contribution of different domains/motifs in the functional diversification of these factors. Guided by the findings of Bacíková and Horowitz (2002); site-specific missense mutants were created in the highly conserved carboxyl-terminus (CR domain and helix 5) of SpPrp18. Additionally, site-specific missense mutants were generated in a conserved amino-terminus domain that is absent in budding yeast Prp18. Our data showed mutants in the highly conserved helix 5 and the CR domain of SpPrp18 to be recessive and non-functional, despite being stably expressed. This contrasts with the temperature-sensitivity conferred by similar mutants in homologous residues in budding yeast Prp18 (Bacíková and Horowitz, 2002). We speculate that the essentiality of the CR domain and helix 5 mutants of SpPrp18 arises due to a defect in spliceosomal association. However, the mutants in conserved residues in the protein’s amino-terminal domain are phenotypically wild type at various growth temperatures tested, suggesting redundant functions for these residues. Our data, based on analysis of a single missense mutant in the highly conserved zinc knuckle motif of SpSlu7, ascribes essential functions for the zinc knuckle motif. We find the mutant to be recessive and non-functional despite stable expression and normal cellular localization of the mutant protein. This contrasts with the behavior of zinc knuckle mutants in budding yeast and human Slu7. Budding yeast Slu7 mutants are functionally wild type and human Slu7 mutants have an altered cellular localization (Frank and Guthrie, 1992; James et al., 2002; Shomron et al., 2004). Possible roles for the zinc knuckle motif of SpSlu7 could be in facilitating interaction of SpSlu7 with U5 snRNA or even with some protein factor. Functional analysis of budding yeast Cef1p-associated complex SpSlu7 and its budding yeast homolog ScSlu7 co-purify with Cdc5/Cef1 in a complex of ~30 proteins together with U2, U5 and U6 snRNAs (Gavin et al., 2002; Ohi et al., 2002). Functional characterization of six proteins of the budding yeast Cef1p complex: Ydl209c (Cwc2/Ntc40), Ycr063w (Cwc14/Bud31), Yju2 (Cwc16), Ygr278w (Cwc22), Ylr424w (Spp382/Ntr1) and Ygl128c (Cwc23) was initiated using a combination of genetic and biochemical approaches. We probed direct protein-protein interactions between members of the Cef1p-associated complex by yeast two-hybrid assays. We also examined the pre-mRNA splicing roles for an essential factor, Yju2/Cwc16 and for a non-essential factor, Ycr063w/Cwc14. Our data reveals direct interaction between Yju2 and early acting factors, Syf1/Ntc90 and Clf1/Ntc77. Similarly interaction of Ydl209c/Cwc2 with early acting splicing factors, Prp19, Syf1/Ntc90 and Clf1/Ntc77 was noted. We created a temperature-sensitive expression strain for YJU2 using a temperature-sensitive Gal4 transcription trans-activator (Chakshusmathi et al., 2004; Mondal et al., 2007) to interrogate the splicing functions of YJU2. RT-PCR and northern blot assays show that depletion of YJU2 causes splicing defects for intron containing pre-mRNAs. We predict early splicing functions for YJU2 as is known for its interacting partners. Furthermore, we find that genetic depletion of the non-essential factor YCR063w causes temperature-sensitivity as has been reported for a few other factors (for e.g. Prp17, Lea1, Snt309/Ntc25, Ecm2) of Cef1p-associated complex (Jones et al, 1995; Chen et al., 1998). Although our yeast two-hybrid data does not reveal any direct interactions between Ycr063w and other proteins of the Cef1p-associated complex, we probed its functions through in vitro splicing assays. Splicing extracts from ycr063w/ycr063w cells show compromised second-step splicing at higher temperatures, thereby implying an auxiliary function for Ycr063w in stabilizing some functionally critical interactions during splicing. These studies employing complementary genetic and biochemical approaches implicate functional divergence of conserved predicted ‘second-step’ fission yeast factors, SpSlu7 and SpPrp18, suggesting co-evolution of splicing factors with changes in genome architecture and intron-exon structure. Our studies on Cef1p-associated complex show novel interactions and implicate pre-mRNA splicing functions for two previously uncharacterized proteins.

Histone deacetylases (HDACs) are a class of enzymes that play a crucial role in DNA expression by removing an acetyl group from the ɛ-N-acetyl lysine residue on histone proteins. Out of 18 isoforms of HDAC enzymes which are classified into 4 classes, only 11 of them are metalloenzymes that require zinc for its catalytic activity. HDACs are considered promising target for drug development in cancer and other parasitic diseases due to their role in gene expression. Histone deacetylase inhibitors (HDACi) can cause cell cycle arrest, and induce differentiation or apotosis. While HDACi shows promising antitumor effects, their mechanism of action and selectivity against cancer cells have not been adequately defined yet. In addition, low oral bioavailability, short half-life time, bone marrow toxicity, and cardiotoxicity limit their use in clinic. Therefore, there is considerable interest in developing compounds with selectivity and specificity towards individual family members of HDACs. The prototypical pharmacophore for HDAC inhibitors consist of a metal-binding moiety that coordinates to the catalytic metal ion within the HDAC active site, a capping group that interacts with the residues at the entrance of the active site and a linker that appropriately positions the metal-binding moiety and capping group for interactions in the active site. It has been shown that modification of cap, cap linking moiety, linker or zinc binding group (ZBG) shows promises of superior potency and isoform selectivity. My thesis research involves manipulating different aspects of the pharmacophoric model to yield not only more potent, selective, and effective drugs but also to help understand the biology of HDAC isoforms. In addition, I was successful in extending studies on HDAC isoforms to other zinc metalloenzymes such as leishmanolysin (gp63) and spliceosome associated zinc-metalloenzymes to understand biology of these zinc metalloenzymes by developing potent and selective small molecule inhibitors. This will aid in improvement of existing therapeutics for treatment of cancer, leishmania, malaria and other genetic disorders.

Alternative splicing provides the cell the ability to generate, from a single gene, multiple protein isoforms, sometimes with different or even antagonistic functions. This process is tightly regulated and alterations in the accurate balance of alternatively spliced mRNAs are a common cause of disease. The main objective of this thesis has been to understand the molecular mechanisms underlying disease-causing defective splicing. Skipping of Fas death receptor exon 6 leads to decreased Fas-ligand induced apoptosis. We have studied how this event is promoted by a mutation at the 3’ splice site and by the proto-oncogene SF2, leading to Autoimmune Lymphoproliferative Syndrome and possibly contributing to tumor progression, respectively. Moreover, we have determined the mechanism by which an antitumor drug, Spliceostatin A, alters 3’ splice site recognition and affects alternative splicing. This thesis underscores the importance of pre-mRNA splicing in disease and how the study of disease-causing aberrant splicing can be used as a tool to understand splicing mechanisms and vice versa. / El processament alternatiu del pre-ARNm proporciona a la cèl•lula l’habilitat de generar, a partir d’un únic gen, proteïnes amb funcions diferents i, fins i tot, antagòniques. Aquest procés està altament regulat i desequilibris en l’abundància de les diferent isoformes són causes comunes de malaltia. L’objectiu principal d’aquesta tesi ha estat entendre el mecanisme molecular a través del qual problemes en el processament del pre-ARNm causen malalties. L’exclusió de l’exó 6 del receptor de mort cel•lular Fas condueix a una disminució de l’apoptosi en resposta al lligand de Fas. Hem estudiat com una mutació al lloc de processament 3’ d’aquest exó i el proto-oncogén SF2 promouen aquest patró, causant el síndrome autoimmune lifoproliferatiu i possiblement contribuint a la progressió tumorogènica, respectivament. A més, hem estudiat el mecanisme pel qual la droga antitumoral Spliceostatin A altera el reconeixement del lloc de processament 3’ i causa canvis en el processament alternatiu de diversos gens. Aquesta tesi posa en evidència la importancia del processament del pre-ARNm en malalties i com l’estudi de mutacions que alteren aquest procés i són causa de malaties pot ser utilitzat con una eina per entendre el mecanisme d’aquest processament i viceversa.