Immediately post-hatch, broiler chicks are exposed to microbes that begin colonizing the gut, including environmental pathogens. One of the costliest enteric diseases in broiler production is necrotic enteritis (NE), caused by the ubiquitous opportunistic bacteria Clostridium perfringens (CP). With the worldwide reduction in antibiotic growth-promoters (AGPs), there is increased interest in natural alternatives to reduce disease and improve broiler health. The overall objective of the studies described herein was to apply probiotics or synbiotics to birds by in ovo application or orally before they leave the hatchery, then evaluate bird performance and various intestinal responses. Data were analyzed in JMP with LS Means to separate means with significance assigned at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. The first 21-day (D) study used 480 male Cobb 500 broilers randomly divided into one of four treatments using a 2x2 factorial design: a no-additive control (CTRL), a one-time oral application of synbiotic at the hatchery fed a basal diet (HS), an oral application of water at the hatchery with dietary synbiotics (DS), and a hatchery synbiotic plus dietary synbiotic (HSDS). Performance was measured on day-of-hatch (DOH), D3, D7, D14, and D21. mRNA abundance of various intestinal markers was measured at D7 and D21, including tight junction proteins ZO-1, ZO-2, and CLD-1; nutrient transporters SGLT1 and PepT1; and immune response markers TLR2, TLR4, and IL-10. HS lowered feed intake (FI) and feed conversion ratio (FCR) without lowering body weight (BW) from D14-21. There was greater abundance of PepT1 mRNA (P ≤ 0.1) and IL-10 mRNA (P ≤ 0.05) on D21 in HSDS. Second, a 21-day pilot study with 480 male and female Cobb 500 broilers was conducted to determine the optimum in ovo dosage level of a probiotic or synbiotic (PROB or SYNB) applied at embryonic day 18 (E18) with subsequent NE challenge using seven treatments: in ovo application of sterile water (CTRL), low (PROB-L or SYNB-L: 1x105 CFU), medium (PROB-M or SYNB-M: 1x106 CFU), or high (PROB-H or SYNB-H: 1x107 CFU) probiotic or synbiotic doses dissolved in sterile water. Performance measurements were taken on DOH, D4, D8, D14 and D21. On D8, NE lesion scores were not impacted by treatment. D8 ileal samples were taken for mRNA abundance of TLR4, IL-10, IL-1β, AvBD8, AvBD10, and AvBD13. SYNB-H had higher abundance of AvBD10 mRNA compared to CTRL (P ≤ 0.1), and higher IL-1β mRNA compared to SYNB-L (P ≤ 0.05). PROB-H and SYNB-H had better performance than the low and medium doses, but were not better than the CTRL. The high doses were chosen for subsequent studies. Third, a longer 42-day study using 1,630 Ross 708 male and female broilers was conducted consisting of the following six treatments. A negative control (NC): sterile water in ovo fed basal corn/soybean meal mash diet without NE challenge; antibiotic growth-promoter (AGP+): sterile water in ovo fed basal diet with virginiamycin (0.5 kg/MT) as an AGP with NE challenge; NC+: same as NC plus NE challenge; SI+: synbiotic in ovo fed the basal diet and NE challenged; SD+: sterile water in ovo fed basal diet supplemented with synbiotic (0.5 kg/MT feed) and NE challenged; and SID+: synbiotic in ovo fed basal diet with synbiotic (0.5 kg/MT feed) with NE challenge. Cumulatively, SID+ had lower FI and FCR than NC+, but no change in BW or BWG. The combination treatment (SID+) often had an additive effect compared to SD+ or SI+ alone on mRNA abundance and D7 cecal fatty acid profiles. SD+ and SID+ also had higher D42 lean:fat ratios compared to NC+. Last, a 42-day study was conducted using 1,630 male and female Ross 708 broilers and the in ovo application of probiotics and subsequent NE challenge with five treatments. NC: sterile water in ovo, fed basal corn/soybean meal mash diet without NE challenge; AGP+: sterile water in ovo, fed basal diet with virginiamycin (0.5 kg/MT of feed) as AGP with NE challenge; NC+: NC treatment, with NE challenge; PI+: probiotic in ovo, fed basal diet, with NE challenge; PD+: sterile water in ovo, fed basal diet supplemented with probiotic (1.3 kg/MT of feed), with NE challenge. The use of probiotics in this study had little effect on performance, lean:fat ratios, and cecal fatty acid profiles, but PD+ increased mRNA abundance of D14 TLR2, D14 TNF-α, and D42 LEAP2 in cecal tonsils compared to controls. PI+ increased mRNA abundance of D7 and D42 MUC2, D7 LEAP2, and D42 TNF-α in the ileum. PI+ increased mRNA abundance in the cecal tonsils of D7 TLR2 and D42 TNF-α. These studies yielded interesting results about probiotics and synbiotics during a NE challenge by evaluating performance, intestinal immune responses, and fatty acid profiles in the ceca of broilers. In conclusion, the probiotic in this study did not improve broiler health during a NE challenge, but synbiotic use in ovo and continuation in the feed showed improvement over in ovo or dietary application alone. Synbiotic improved FCR over a challenged control, and altered mRNA abundance in the small intestine. / Doctor of Philosophy / The poultry industry is one of the most popular animal protein sources worldwide. As with any livestock operation, industry goals include optimizing animal health and well-being, maximizing animal productivity, and producing quality products in the most cost effective manner. Improvements in genetics, nutrition, and management have increased productivity and cut costs. One important application was the low-level use of antibiotics in feed. These medications reduced the risk of disease outbreak in flocks, which led to healthier birds and improved growth rates. However, when global concern of antibiotic resistance in human medicine came to light, both the livestock industry and governing bodies implemented voluntary and mandatory reduction or elimination of antibiotics. Previously, these important antibiotics helped to control costly diseases. As they are removed, alternatives to antibiotics will be important in disease control and prevention. A major group of alternatives to antibiotics in poultry includes probiotics, prebiotics, and synbiotics. Probiotic bacteria are considered 'good bacteria' in the gut, and provide various health benefits to the host. Prebiotics are non-living substances that support the growth of healthy bacteria. A synbiotic is the combination of both probiotics and prebiotics in a single application method. The goal of this research project was to give probiotics or synbiotics to broiler chicks and evaluate their potential benefits and effects on bird performance and the immune response. Ideally, applying probiotic bacteria as early as possible might translate into early colonization of the gut with healthy bacteria. This included oral application of synbiotics at the hatchery, or by safely injecting them into part of the egg that is swallowed by the chick embryo before hatch. This egg application, or in ovo application, is a safe, effective, widely-practiced method of vaccinating chicks to jumpstart their defense against disease. By vaccinating them in ovo, they can start to prime the immune system before they even hatch. Applying probiotics in ovo may improve health after early gut colonization with beneficial microbes. Numerous studies on natural alternatives to antibiotics have been conducted, with varying results. Results of this research indicate that in ovo application of probiotics and synbiotics is safe. Birds that received probiotics in the feed often performed similar to those that received none. However, the in ovo use of synbiotics combined with the continued use in the feed after hatch improved efficiency in broilers during an intestinal disease challenge and improved various aspects of gut function. Overall, as antibiotics are phased out, using probiotics and synbiotics may improve poultry health, but continued research will help understand the optimum ways to use them.

The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC-independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation. / Ph. D.

Nescient Helix Loop Helix 2 (Nhlh2) is a member of the basic helix-loop-helix transcription factor family. Mice with a targeted deletion of Nhlh2, called N2KO mice, show adult onset obesity in both males and females. Nhlh2 regulates other genes by binding to the E-box in the promoter region of these genes. This transcription factor regulates many other transcription factors including MC4R and PC1/3 which are associated with human obesity. The Nhlh2 promoter has been analyzed for putative transcription factors binding sites. These putative binding sites have been tested to be the regulators of Nhlh2 by transactivation assays with mutant promoters, Electrophoretic Shift Assay (EMSA), and Chromatin Immunoprecipitation Assay (ChIP) as methods to investigate the DNA-protein binding. The results of these experiments showed that the Nhlh2 promoter has five Signal Transducer and Activator of Transcription 3 (Stat3) binding site motifs at -47, -65, -80, -281, -294 and two Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB) binding site motifs at -67 and -135. While NFκB acts as a negative regulator of Nhlh2, this research showed that Stat3 acts as a regulator for the Nhlh2 basal expression and leptin stimulation. The ChIP assay using chromatin from mouse hypothalamus and antibodies against Stat3 and the NFκB subunits P50, P65, and c-Rel demonstrated that all of these antibodies were able to pull down the part of the Nhlh2 promoter containing the binding sites of Stat3 and NFκB. The EMSA results not only demonstrated that NFκB and Stat3 binding site motifs are real binding sites, but also exists the possibility of a relationship between these transcription factors to regulate Nhlh2 expression with leptin stimulation. An effort in analyzing the human NHLH2 3'UTR showed that one of the SNPs located at position 1568 in the NHLH2 mRNA (NHLH2A^1568G) which converts adenosine to guanine might have the potential to decrease the mRNA stability. For more investigation about this SNP, the mouse Nhlh2 tail was cloned into 2 different vectors and these vectors were subjected to site directed mutagenesis to create the 3'UTR SNP that convert A to G. One of these vectors used luciferase as a reporter gene for expression while the other one was used to measure Nhlh2 mRNA stability. These vectors were transfected into hypothalamic cell line N29/2 to test the effect of this SNP on Nhlh2 expression. This study demonstrated that this SNP down regulated luciferase expression and also decreased Nhlh2 mRNA stability. Taken together, this study demonstrated that Nhlh2 could be regulated transcriptionally by both NFκB and Stat3 transcription factors and post-transcripitionally by the 3'UTR SNP that converts adenosine to guanine. / Ph. D.

The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications. / Ph. D.

Spinal muscular atrophy (SMA) is a devastating disorder caused by deletions and mutations in the survival of motor neuron (SMN1) gene and is marked by motor neuron loss and muscle weakness. While its genetic basis is clear, the underlying molecular mechanisms remain elusive. Decreased levels of the survival of motor neuron (SMN) protein, encoded by the SMN1 gene, are implicated in SMA pathology. Despite splicing has been under the spotlight as a major mechanism impaired in SMA, recent evidence suggests that SMN deficiency also disrupts protein translation in vivo in a mouse model of severe SMA, complicating SMA's molecular landscape. This thesis examines the impact of SMN protein loss on translation in SMA mouse models across tissues, post-natal and pre-natal disease stages, focusing on both mild (Chapter 1) and severe forms of SMA (Chapter 2) respectively. To tackle this question, in this thesis, I took advantage of multiple cutting-edge and sequencing-based techniques (ribosome profiling and RNA-seq) coupled with biochemical and molecular biology-based assay (polysome profiling, co-sedimentation profiles, qPCR, and western blotting), which applied to study in molecular detail the translational defects in the brain, spinal cord and liver at asymptomatic, pre-symptomatic and early symptomatic stages of SMA. Polysome profiling in control mice (Smn2B/+) reveals a gradual increase in SMN association with ribosomes/polysomes during postnatal development, indicating dynamic SMN function in protein translation during post-natal development. In SMA condition, where SMN protein levels drop, this binding reveals a tissue-specific decrease in the spinal cord and liver. Through ribosome profiling, numerous alterations in translation were identified at the pre-symptomatic stage of the disease, suggesting that translational defects are features of the early stages of SMA. Importantly these alterations are independent of transcriptional and splicing changes. Although no gene was found to be in common, I found that genes altered in at least 2 tissues are involved in the same processes. The dysregulated mRNAs exhibit rare codons at the beginning of coding sequences in all three tissues, as shown in the case of the severe model of SMA. 4 From these common processes I have identified specific mRNA targets that play key roles in the organization of the extracellular matrix I validated the presence of translational changes in Col1a1, Col1a2, and Spp1 highlighted effects of SMN deficiency on translational regulation, in the absence of transcriptional alterations. Validation studies in both mice and SMA patient-derived fibroblasts further underscored the potential of translational dysregulation and drop in Col1a1 protein expression during SMA progression. Finally, prenatal studies have revealed distinct translational changes in embryonic tissues from Taiwanese mice. Despite no alterations in global translation, a drop in SMN association with ribosomes/polysomes and tissue-specific differences in ribosome occupancy were observed. Also, in this case, dysregulated mRNAs exhibit rare codons at the beginning of coding sequences. These findings shed light on the unique molecular landscape of prenatal development in the context of SMN deficiency. In summary, this study provides insights into translation dysregulation in SMA pathology, emphasizing tissue-specific effects and developmental stage-dependent alterations. By elucidating the complex relationship between SMN protein function and translational dynamics, it lays the groundwork for targeted therapeutic strategies and biomarkers to improve SMA management. Ongoing investigations into prenatal development and translation dynamics are crucial for a comprehensive understanding of SMA pathogenesis and effective treatment development.

No / Skin development, postnatal growth and regeneration are governed by complex and well-balanced programs of gene activation and silencing. The crosstalk between small non-coding microRNAs (miRNAs) and mRNAs is highly important for steadiness of signal transduction and transcriptional activities as well as for maintenance of homeostasis in many organs, including the skin. Recent data demonstrated that the expression of many genes, including cell type-specific master transcription regulators implicated in the control of skin development and homeostasis, is regulated by miRNAs. In addition, individual miRNAs could mediate the effects of these signaling pathways through being their downstream components. In turn, the expression of a major constituent of the miRNA processing machinery, Dicer, can be controlled by cell type-specific transcription factors, which form negative feedback loop mechanisms essential for the proper execution of cell differentiation- associated gene expression programs and cell-cell communications during normal skin development and regeneration. This review summarizes the available data on how miRNA/mRNA regulatory networks are involved in the control of skin development, epidermal homeostasis, hair cycle-associated tissue remodeling and pigmentation. Understanding of the fundamental mechanisms that govern skin development and regeneration will contribute to the development of new therapeutic approaches for many pathological skin conditions by using miRNA-based interventions.

Tesis por compendio / [EN] One of the defining features of a eukaryotic cell is the presence of a nuclear envelope. This allowed the physical separation between nucleus and cytoplasm, although the presence of a variable number of openings called nuclear pore complexes (NPCs) allowed a constant flow of molecules and information between the two compartments. Certain molecules passively diffuse, but others need energy and specific interactions with transporters and components of the NPC to travel between through both compartments. The messenger RNAs (mRNAs) are among the molecules selectively exported from the nucleus to the cytoplasm. The physical separation between nucleus and cytoplasm isolates the processes of transcription and translation in eukaryotic cells, allowing the cell to select core transcripts competent for export and that will lead to a functional protein in the cytoplasm. The right transcript levels in a cell, depending on the nutritional requirements, reproductive or relationship with the environment is essential for life. To this end, mechanisms regulating transcription, processing, stability, degradation, export or translation of the transcripts, are physically and spatially highly coupled in order to finely regulate the transcript levels in the cell. In Saccharomyces cerevisiae, SUS1 codes for a small protein of 11 kDa highly conserved in all eukaryotes. SUS1 is part of the SAGA transcriptional co-activator, being a submodule component involved in chromatin remodeling. In addition, SUS1 is one of the components of the TREX2 complex, wich interacts with the nuclear pore in the periphery of the nucleus and it is involved in the export of messenger RNAs. The presence of SUS1 in both complexes allows the physical and spatial coupling phenomena of transcription and export of mRNAs. In addition, SUS1 has two introns which is an unusual fact for the S. cerevisiae genome. Unlike other fungi or metazoans, the percentage of genes with introns in S. cerevisiae is very low (5%) and only 10 genes have more than one intron interrupting its coding sequence. The unusual characteristics of SUS1, the role of Sus1 coordinating processes during mRNA biogenesis and its functional conservation in higher eukaryotes, led the research conducted in this dissertation. In this work we studied in detail the biogenesis of SUS1 transcripts. We have identified different factors, acting in cis and trans that are involved in regulating the expression of SUS1 and function of the protein it encodes. On the other hand, we have studied the genetic relationship of SUS1 with components of the 5 '- 3' cytoplasmic degradation machinery and expanded the knowledge about the role of SUS1 during the biogenesis of mRNAs, not only in the nucleus but also in the cytoplasm. / [ES] Una de las características que definen a una célula eucariota es la presencia de una envoltura nuclear. Aunque este hecho permite la separación física entre núcleo y citoplasma, la presencia de un número variable de aberturas, denominadas complejos del poro nuclear (NPCs), permiten un flujo constante de moléculas e información entre ambos compartimentos. Ciertas moléculas difunden de forma pasiva, pero otras necesitan energía e interacciones específicas a través de transportadores y componentes del NPC para transitar entre ambos compartimentos. Entre las moléculas selectivamente exportadas del núcleo al citoplasma se encuentran los RNAs mensajeros (mRNAs). La separación física del núcleo y del citoplasma en eucariotas, aísla los procesos de transcripción y traducción, permitiendo a la célula seleccionar en el núcleo los transcritos competentes para ser exportados y que darán lugar a una proteína funcional en el citoplasma. Adecuar los niveles de transcritos en una célula, en función de las necesidades nutritivas, reproductivas o de relación con el entorno, es esencial para la vida. Para ello, los mecanismos encargados de regular la transcripción, el procesamiento, la estabilidad, la degradación, el exporte o la traducción de los transcritos, se encuentran altamente acoplados física y espacialmente con el fin de regular finamente los niveles de mensajeros en la célula. En el núcleo de las células de la levadura Saccharomyces cerevisiae se encuentra Sus1, una proteína de 11 kDa altamente conservada en eucariotas. Sus1 forma parte del co-activador transcripcional SAGA, siendo componente de un submódulo implicado en la desubicuitinación de la histona H2B. Además, Sus1 es uno de los componentes del complejo TREX2, que interacciona con el poro nuclear en la periferia del nucleo y está implicado en el exporte de RNAs mensajeros y en estabilidad genómica. La presencia de Sus1 en ambos complejos permite el acoplamiento físico y espacial de los fenómenos de transcripción y exporte de mRNAs. Además, el gen SUS1 posee dos intrones, siendo este un evento muy inusual en el genoma de S. cerevisiae. A diferencia de otros hongos o metazoos, el porcentaje de genes con intrones en S. cerevisiae es muy reducido (5%) y solo 10 genes poseen más de un intrón interrumpiendo su secuencia codificante. Las características peculiares del gen SUS1, el papel de la proteína que codifica coordinando procesos durante la biogénesis del mRNA y su conservación funcional en eucariotas superiores, motivó las investigaciones llevadas a cabo en esta tesis doctoral. En este trabajo hemos estudiado en detalle la biogénesis de los transcritos de SUS1. Se han identificado diferentes factores, tanto en cis como en trans, implicados en la regulación de la expresión de SUS1 y en la función de la proteína que codifica. Por otro lado, hemos estudiado la relación genética de SUS1 con componentes de la maquinaria de degradación citoplasmática 5'-3' y hemos ampliado los conocimientos respecto al papel de Sus1 durante la biogénesis de los mRNAs, no solo en el núcleo sino también en el citoplasma. / [CA] Una de les característiques que definixen a una cèl·lula eucariota és la presència d'un embolcall nuclear. Este fet permet la separació física entre nucli i citoplasma, encara que la presència d'un nombre variable d'obertures, denominades complexos del porus nuclear (NPCs), permet un flux constant de molècules i informació entre ambdós compartiments. Certes molècules difonen de forma passiva, però altres necessiten energia i interaccions específiques amb transportadors i components del NPC per a transitar entre ambdós compartiments. Entre les molècules selectivament exportades del nucli al citoplasma es troben els RNAs missatgers (mRNAs). La separació física del nucli i del citoplasma aïlla els processos de transcripció i traducció en eucariòtes, permetent a la cèl·lula seleccionar en el nucli els transcrits competents per a ser exportats i que donaran lloc a una proteïna funcional en el citoplasma. Adequar els nivells de transcrits en una cèl·lula, en funció de les necessitats nutritives, reproductives o de relació amb l'entorn, és essencial per a la vida. Per a això, els mecanismes encarregats de regular la transcripció, el processament, l'estabilitat, la degradació, l'exportació o la traducció dels transcrits, es troben altament acoblats física i espacialment amb el fi de regular finament els nivells de missatgers en la cèl·lula. En el nucli de les cèl·lules del rent Saccharomyces cerevisiae es troba Sus1, una xicoteta proteïna d'11 kDa altament conservada en eucariotes. Sus1 forma part del coactivador transcripcional SAGA, sent component d'un submòdul implicat en la modificació de histones. A més, Sus1 és un dels components del complex TREX2, que interacciona amb l'embolall nuclear a la perriferia del nucli i està implicat en l'export de RNAs missatgers. La presència de Sus1 en ambdós complexos permet l'adaptament físic i espacial dels fenòmens de transcripció i exportació de mRNAs. A més, el gen SUS1 posseïx dos introns i este fet és inusual en el genoma de S. cerevisiae. A diferència d'altres fongs o metazous, el percentatge de gens amb introns en S. cerevisiae és molt reduït (5%) i només 10 gens posseïxen més d'un intró interrompent la seua seqüència codificant. Les característiques peculiars del gen SUS1, el paper de la proteïna que codifica coordinant processos durant la biogènesi del mRNA i la seua conservació funcional en eucariotes superiors, va motivar les investigacions dutes a terme en esta tesi doctoral. En este treball hem estudiat en detall la biogènesi dels transcrits de SUS1. S'han identificat diferents factors, tant en cis com en trans, implicats en la regulació de l'expressió de SUS1 i en la funció de la proteïna que codifica. D'altra banda, hem estudiat la relació genètica de SUS1 amb components de la maquinària de degradació citoplasmática 5'-3' i hem ampliat els coneixements respecte al paper de Sus1 durant la biogènesi dels mRNAs, no sols en el nucli sinó també al citoplasma. / Cuenca Bono, B. (2016). Identificación y caracterización de elementos reguladores de la expresión de SUS1 y nuevas funciones celulares para la proteina Sus1 en Saccharomyces cerevisiae [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62327 / Compendio

Post-transcriptional control of gene expression in Eukaryotes plays a pivotal role in determining intricated networks defining physiological and pathological conditions among each organism. RNA Binding Proteins (RBPs), by exploiting RNA-protein and protein-protein interactions, have been recognized as the main actors in modulating these processes. As a consequence, RBPs aberrant expression, modulation or mis-localization, leads to the insurgence of complex phenotypes and diseases. Therefore, targeting and modulating the activity of RBPs found associated to different pathologies represents a new promising therapeutic strategy. During my PhD I aimed at identify, characterize and refine inhibitors targeting the RNA binding protein HuR. HuR belongs to the ELAVL protein family, it is ubiquitously expressed in the cells and among tissues and highly conserved throughout mammalian evolution. By binding AU/U rich elements (ARE) in the 3’UTRs of mRNAs, HuR mainly stabilizes its target transcripts, enhancing their translation. ARE sequences are found in 7% of the human mRNAs, coding for protein involved in key cellular processes as: immune response and inflammation, cell division and proliferation, angiogenesis, senescence and apoptosis. Hence, dysregulation in HuR expression and in its subcellular localization have been associated with the insurgence of several pathologies, mostly cancers and inflammation diseases. Notably, malignant transformations and poor prognosis in patients have been found characterized by highly nuclear or cytosolic HuR expression in a significant number of human cancers. Indeed, the majority of HuR regulated transcripts encode for protein responsible for the appearance of several cancerogenic traits. In particular, critical crosstalk established between cancer cells and inflammation processes play a pivotal role in worsening and compromising cancers development and onset. Moreover, considering that 90% of mRNAs coding for cytokines and chemokines contains repeated AREs sites in the 3’UTR, HuR plays a strong regulatory role in immune system (innate and adaptive) development and homeostasis as well as in pathogenic mechanisms. The searching for HuR inhibitors represents a challenging area, in the drug discovery field, due to its pleiotropic functions and its intrinsic structural complexity, which presents unfolded regions and sequences prone to aggregation. HuR disruptors have been reported in the literature, but without systematic studies, thus the identification of a new class of small molecules is still at the beginning. Among the molecules discovered so far, in 2015 our group identified through a High-throughput Screening a natural compound, DHTS, as a bona fide HuR inhibitor. Following that finding, we, me included, ascribed to the molecule a well-defined mechanism of action, identifying the specific binding sites on which HuR:DHTS interaction is based, defining that upon the mRNA binding DHTS interplays with HuR maintaining the protein in a closed conformation, thus inhibiting its function. Furthermore, we demonstrated DHTS anti-cancer activity in vitro, in cellular context and in vivo, in an HuR-dependent manner. In this way, DHTS represented the molecular scaffold, for the generation of a new class of highly potent HuR inhibitors, called Tanshinone Mimics (TMs). A functional oriented approach was applied for the synthesis of new molecules harboring only DHTS chemical elements responsible for HuR targeting, leading to a completely new molecular scaffold, not previously described in the literature, with respect to the ancestor molecule. I have characterized and identified more potent molecules, describing their anticancer properties, through the evaluation of their capabilities of downregulating the total expression level of well-known HuR targets, coding for proteins involved in tumor insurgence and progression, as VEGF, ERBB2 and CTNNB1, and reducing cancer cell migration, cell cycle progression in a minor extent. On the other end, I have explored TMs anti-inflammatory properties, counteracting the inflammatory response mediated by macrophages, directly impairing the binding between HuR and its pro-inflammatory targets, diminishing their expression and related protein secretion. Moreover, I have put evidences on TMs activity in vivo in acute inflammation mouse models. Lastly, I have evaluated TMs activity in affecting T-cells proliferation, on which HuR it is known to play a regulatory role. In conclusion, we identified TMs with Structure-Activity Relationships (SARs) towards HuR inhibition and its biological implications, aimed at ameliorating their specificity and bioavailability suitable for in vivo therapeutic strategies.

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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