The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models. Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD. Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.

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For many human protein-coding genes, alternative cleavage and polyadenylation (APA) of pre-mRNA generates distinct 3' untranslated regions (3'UTRs) with differing regulatory potential. Widespread 3'UTR shortening via APA occurs in proliferative cell states, including cancer, where it can lead to oncogene overexpression. There has therefore been significant interest in identifying factors which influence poly(A) site choice in different physiological states. The multi-subunit human cleavage factor I complex (CFIm), a core component of the mammalian pre-mRNA cleavage machinery, has been identified as a potential master regulator of APA, as its depletion leads to widespread 3'UTR shortening. However, mechanistic understanding of how CFIm influences poly(A) site selection, and how its activity is regulated, is lacking. In this work, gene editing was used to generate cell lines with substantial, permanent depletion of the 25 kDa or 68 kDa subunits of CFIm (CFIm25 and CFIm68), which exhibited the expected 3'UTR shortening for representative transcripts. Reversal of this 3'UTR shortening by CFIm25 or CFIm68 re-expression provided the basis for a complementation assay, which allowed various aspects of CFIm25 and CFIm68 function to be investigated in vivo. The capacity of CFIm25 to recognise UGUA RNA sequences was shown to make an important contribution to poly(A) site selection transcriptome-wide, and a novel function for the C-terminal arginine/serine-rich (RS domain) of CFIm68 in poly(A) site selection was identified. The potential contribution of CFIm post-translational modification (PTM) to APA regulation was also explored. Novel acetylation sites on CFIm25 and CFIm68 were identified, as well as extensive serine phosphorylation in the CFIm68 RS domain. Complementation analysis revealed that phosphomimetic mutations in this RS domain inhibited distal poly(A) site selection, suggesting a potential role for CFIm68 phosphorylation in APA regulation. Taken together, the findings presented here provide insights into several important determinants of CFIm function, and the complementation assay developed provides a useful tool for future investigations.

Made available in DSpace on 2018-08-02T00:16:24Z (GMT). No. of bitstreams: 1 tese_6085_IRANY RODRIGUES PRETTI.pdf: 1820028 bytes, checksum: 206c5b4515936d11e0d571040d9689c8 (MD5) Previous issue date: 2012-02-29 / RESUMO O maracujazeiro é cultivado em quase todo o território brasileiro. O Estado do Espírito Santo atualmente ocupa o quarto lugar em produção no país. A produção capixaba, de Passiflora edulis, está em expansão, tendo em vista que nos últimos anos houve um aumento na área plantada de maracujazeiro de 500 ha, em 1990, para 2.429 ha, em março de 2008, atingindo em dezembro de 2010, 2.800 ha plantados. Contudo, a maior parte da produção capixaba ainda se concentra na região norte do Estado, com destaque para os municípios de Jaguaré e Sooretama. Apesar dos números mostrarem avanços na produção dessa frutífera, o Espírito Santo é caracterizado por solos ácidos e alto teor de alumínio. Dentre as respostas desencadeadas pela exposição ao alumínio estão: inibição do crescimento radicular, inibição da divisão celular, deficiência de nutrientes, além da ativação de rotas de sinalização e alterações em nível de proteínas e transcritos de RNA. Dessa forma, torna-se fundamental o estudo de componentes do sistema de defesa antioxidativo do maracujazeiro cultivado sob exposição ao alumínio. As EROs podem representar grave ameaça à célula, sendo uma das respostas aos estresses ambientais, como a toxidez por alumínio. Contudo, poucos trabalhos têm relatado os efeitos do Al na parte aérea da planta. Por isso, o objetivo deste trabalho foi verificar a resposta o sistema antioxidante de P.edulis sob tais condições. O estudo do estresse oxidativo induzido por alumínio demonstrou que a expressão do gene Cat e Sod foi aumentada nas plantas em solução nutritiva, e somente na cv. FB100 esse aumento foi acompanhado pelo incremento da atividade enzimática de SOD, o que indica maior eficiência desta na remoção de EROs. As plantas cultivadas em campo demonstraram que a expressão das enzimas antioxidantes na lavoura com alto teor de alumínio não estava ativada no momento da coleta, apesar da elevada atividade de SOD e APX. Esta resposta possivelmente foi determinada pelo tempo prolongado ao qual as plantas estavam expostas ao estresse.

Abstract In malignancies many alterations involving matrix macromolecule synthesis, secretion and assembly into basement membranes (BMs) as well as their degradation are present. The most important groups associated with matrix turnover are matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). In this study altogether 285 tissue samples were investigated comprising various malignant epithelial tumors and normal tissue structures, in which the distribution of different laminin chains was studied immunohistochemically. Laminin α5, β1 and γ1 were detected almost in all the BMs studied including normal tissues and malignancies, whereas α1 chain of laminin was present only in certain BMs. Laminin γ2 chain was solely expressed by epithelial BMs and was present in intracellular space especially in individual carcinoma cells infiltrating in the tumor stroma and in tumor cells in close contact with BM zone. Generally epithelial tumors contained quite well-formed BMs around their tumor clusters, except for infiltrative breast carcinoma and diffuse type gastric carcinoma. In situ hybridization revealed that only epithelial cells contained mRNAs for laminin α1 and γ2 chains, whereas laminin β1 chain and α1(IV) collagen were synthesized mainly by stromal cells. mRNA for MMP-2 was produced mainly by stromal cells in hepatocellular carcinoma of liver (HCC) and pancreatic adenocarcinoma, whereas MMP-9 and MT1-MMP were equally synthesized by carcinoma cells and cells of tumor stroma. However, in HCCs of grade III carcinoma cells predominated in their MT1-MMP expression. All three MMPs were immunolocalized to malignant epithelial cells and showed variably stromal cell positivity. Statistically mRNA synthesis for MT1-MMP was significantly associated with the shortened survival of patients with HCC (P ≤ 0.01). TIMP-1-3 mRNA, and especially TIMP-3, expressions in normal endometrium were significantly increased in endometrial stromal cells towards the secretory phase. In various endometrial hyperplasias TIMPs and MT1-MMP expressions were quite comparable to those seen in proliferating endometrium. In endometrial adenocarcinomas their expressions were significantly increased and the most intensified mRNA expressions were seen in grade III adenocarcinomas. Especially TIMP-3 and MT1-MMP mRNAs were synthesized by carcinoma cells. The results indicate that epithelial malignancies are capable of active synthesis and assembly of BM macromolecules. Simultaneous matrix synthesis and degradation seen in malignancies suggest that the mechanisms involved in matrix turnover are not lost during malignant transformation. mRNA synthesis for MMPs and TIMPs is generally increased in epithelial malignancies. The results therefore strongly support the concept that MMPs have an active role in carcinoma cell invasion.

The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models. Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD. Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.

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.

Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. The SUS1 gene of Saccharomyces cerevisiae is unusual, as it contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. In this thesis project, we have investigated whether the structure adopted by SUS1 RNA sequences contributes to regulate the splicing of this gene. Using in silico analyses together with NMR spectroscopy, gel electrophoresis and UV thermal denaturation experiments, we first show that the downstream intron (I2) of SUS1 forms a weakly-stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semi-quantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild-type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. The second part of the thesis project focuses on the exon located between the two introns of the SUS1 gene. This middle exon (E2) can be skipped during splicing, is generated in circular form, and has been found to influence the splicing of the flanking introns, an unusual situation in budding yeast where splicing mainly relies on intron recognition. Using NMR spectroscopy, gel electrophoresis, UV thermal denaturation and ribose 2'-OH modification experiments combined with computational predictions, we show that E2 of SUS1 comprises a conserved double-helical stem topped by a three-way junction. One of the hairpins emerging from the junction exhibited significant thermal stability and was closed by an unusually structured purine-rich loop. This loop contained two consecutive sheared G:A base pairs and was structurally related to the substrate loop of the VS ribozyme. Cellular assays revealed that three mutants containing altered E2 structures had impaired SUS1 expression and that a compensatory mutation restoring the conserved stem recovered expression to wild-type levels. Semi-quantitative RT-PCR experiments indicated that all mutants were capable of altering the quantities of unspliced and/or fully-spliced SUS1 RNA transcripts relative to wildtype. Overall, the results gathered in this thesis project indicate that RNA structures formed by the middle exon and the second intron of the S. cerevisiae SUS1 gene are relevant for splicing and also influence other processes of SUS1 mRNA biogenesis. / Sus1 es una proteína conservada implicada en remodelación de cromatina y biogénesis de moléculas de ARNm. El gen SUS1 de Saccharomyces cerevisiae es peculiar, ya que contiene dos intrones y sufre un proceso de ayuste (corte y empalme) alternativo, reteniendo uno o ambos intrones en respuesta a cambios en las condiciones ambientales. El ayuste del ARNpre-m de SUS1 puede permitir a la célula controlar la expresión de la proteína Sus1, pero los mecanismos que regulan este proceso son poco conocidos. En este proyecto de tesis hemos investigado si la estructura adoptada por secuencias de ARN de SUS1 contribuye a regular el proceso de ayuste de este gen. Utilizando análisis in silico junto con espectroscopia de RMN, electroforesis en gel y experimentos de desnaturalización térmica monitorizados por UV, primero demostramos que el ARN del segundo intrón (I2) del gen SUS1 forma una horquilla débilmente estable de 37 nucleótidos. Esta horquilla contiene nucleótidos del sitio de ramificación (branch site) en su bucle apical y nucleótidos del sitio 3' de empalme adyacentes al extremo inferior del tallo. A través de ensayos funcionales descubrimos que dos de cuatro mutantes que alteran la estructura de la horquilla I2 exhibían peor expresión de SUS1. Experimentos de RT-PCR semicuantitativos indicaron que todos los mutantes acumularon ARNpre-m SUS1 no ayustado y/o indujeron cambios en los niveles de ARNm maduro con respecto a la secuencia silvestre. Además, las funciones celulares de Sus1 relativas a desubicuitinación de histona H2B y transporte de ARNm se vieron afectadas en los mutantes de la horquilla I2 que inhibían el proceso de ayuste. La segunda parte de la memoria de tesis se centra en el análisis del exón central (E2) situado entre los dos intrones del gen SUS1. Este exón puede eliminarse durante el proceso de ayuste, se genera en forma circular, e influye en el procesamiento de los intrones adyacentes, una situación inusual para las regiones exónicas de S. cerevisiae, donde el ayuste se basa principalmente en el reconocimiento de intrones. Utilizando experimentos de espectroscopía de RMN, electroforesis en gel, desnaturalización térmica y modificación química combinados con predicciones computacionales, demostramos que el ARN del exón E2 de SUS1 forma un tallo conservado de doble hélice coronado por una intersección de tres hélices. Una de las horquillas que emergen de esta intersección presentó una estabilidad térmica significativa, así como un bucle apical rico en purinas inusualmente estructurado. Este bucle contiene dos pares de bases G:A consecutivos y está estructuralmente relacionado con el bucle de substrato de la ribozima VS. Ensayos celulares revelaron que tres mutantes con estructuras modificadas de E2 exhibían peor expresión de SUS1, y que una mutación compensatoria que restauraba el tallo conservado recuperaba la expresión a los niveles de la secuencia silvestre. Experimentos de RT-PCR semicuantitativos indicaron que todos los mutantes de E2 eran capaces de alterar las cantidades de transcritos ayustados y no ayustados de SUS1 con respecto a la secuencia silvestre. En general, los resultados obtenidos en este proyecto de tesis indican que las estructuras de ARN formadas por el exón central y el segundo intrón del gen SUS1 de S. cerevisiae son relevantes para el ayuste y otros procesos implicados en la biogénesis del ARNm del gen SUS1. / Sus1 és una proteïna conservada implicada a la remodelació de la cromatina i la biogènesi de l'ARNm. El gen SUS1 de Saccharomyces cerevisiae és inusual, ja que conté dos introns i s'empalma de manera alternativa, retenint un o ambdós introns en resposta a canvis en les condicions ambientals. L'empalmament de SUS1 pot permetre a la cèl·lula controlar l'expressió de Sus1, però els mecanismes que regulen aquest procés són segueixen sent desconeguts. En aquest projecte de tesi investiguem si l'estructura adoptada per seqüències d'ARN de SUS1 contribueix a regular l'empalmament d'aquest gen. Emprant anàlisi in silico juntament amb espectrometria de RMN, electroforesi en gel i experiments de desnaturalització tèrmica d'UV, es mostra primer que l'intró aigües a baix (I2) de SUS1 forma una estructura de forqueta de 37 nucleòtids feblement estable que conté el lloc de la branca a prop del seu bucle apical; i el lloc d'empalmamnet 3¿ després de l'extrem de la forqueta. Un assaig cel·lular va revelar que dos de quatre mutants que contenien estructures alterades de l'I2 havien modificat significativament l'expressió de SUS1. Els experiments semi-quantitatius de RT-PCR van indicar que tots els mutants acumulaven el pre-ARNm madur respecte al tipus salvatge. Concomitantment, les funcions cel·lulars de Sus1 a la desubiqüitinació de la histona H2B i l'exportació d'ARNm es van veure afectats als mutants de la forqueta d'I2 que inhibeixen l'empalmament. La segona part del projecte de tesi se centra a l'exó situat entre els dos introns del gen SUS1. Aquest exó (E2) es pot ometre durant l'empalmament, es genera amb forma circular, i s'ha trobat que influeix a l'empalmamet dels introns que flanquegen, una situació inusual al llevat on l'empalmament està basat principalment al reconeixement d'introns. Emprant espectroscòpia de RMN, electroforesi en gel, desnaturalització tèrmica d'UV i experiments de modificació de ribosa 2¿-OH combinats amb prediccions computacionals, mostrem que E2 de SUS1 comprén un tall conservat de doble hèlix corornat per una unió de tres vies. Una de les forquetes que emergeixen de la unió, va mostrar una estabilitat tèrmica significativa i va ser tapada per un bucle ric en purina inusualment estructurat. Aquest bucle contenia dos pars de bases G:A tallats consecutivament i estava estructuralment relacionat amb el bucle de substrat del ribozim VS. Els assajos cel·lulars van revelar que tres mutants que contenien estructures alterades de E2 havien alterat l'expressió de SUS1 i que una mutació compensatòria que restaurava el tall conservat recuperava l'expressió a nivells del tipus salvatge. Els assajos cel·lulars van revelat que tres mutants que contenien estructures alterades d'E2 havien alterat l'expressió de SUS1 i que una mutació compensatòria que restaurava el tall conservat recuperava l'expressió a nivell d'un tipus salvatge. Els experiments semi-quantitatius de RT-PCR van indicar que tots els mutants eren capaços d'alterar les quantitats de transcrits d'ARN de SUS1 no empalmats i/o empalmats en relació amb el tipus salvatge. En general, els resultats obtinguts en aquesta investigació indiquen que les estructures d'ARN formades per l'exó mitjà i el segon intró de SUS1 de S. cerevisiae són rellevants per l'empalmament i també influeixen a altres processos de biogènesi de l'ARN de SUS1 / Abuqattam, AN. (2017). IDENTIFICATION OF RNA STRUCTURES MODULATING THE EXPRESSION OF THE mRNA BIOGENESIS FACTOR SUS1 [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90524 / TESIS

Endocannabinoid signaling is well studied in mammals and known to be involved in numerous pathological and physiological processes. Fatty acid amide hydrolase (FAAH) terminates endocannabinoid signaling in mammals. In Physcomitrella patens, we identified nine orthologs of FAAH (PpFAAH1 to PpFAAH9) with the characteristic catalytic triad and amidase signature sequence. Kinetics of PpFAAH1 showed specificity towards anandamide (AEA) at 37°C and pH 8.0. Further biophysical and bioinformatic analyses revealed that, structurally, PpFAAH1 to PpFAAH4 were closely associated to the plant FAAH whereas PpFAAH6 to PpFAAH9 were more closely associated to the animal FAAH. A substrate entry gate or ‘dynamic paddle’ in FAAH is fully formed in vertebrates but absent or not fully developed in non-vertebrates and plants. In planta analysis revealed that PpFAAH responded differently with saturated and unsaturated N-acylethanolamines (NAEs). In vivo amidohydrolase activity showed specificity associated with developmental stages. Additionally, overexpression of PpFAAH1 indicated the need for NAEs in developmental transition. To understand and identify key molecules related to endocannabinoid signaling in P. patens, we used high-throughput RNA sequencing. We analyzed temporal expression of mRNA and long non-coding RNA (lncRNA) in response not only to exogenous anandamide but also its precursor arachidonic acid and abscisic acid (ABA, a stress hormone). From the 40 RNA-seq libraries generated, we identified 4244 novel lncRNAs. The highest number of differentially expressed genes (DEGs) for both mRNA and lncRNA were detected on short-term exposure (1 h) to AEA. Furthermore, gene ontology enrichment analysis showed that 17 genes related to activation of the G protein-coupled receptor signaling pathway were highly expressed along with a number of genes associated with organelle relocation and localization. We identified key signaling components of AEA that showed significant difference when compared with ABA. This study provides a fundamental understanding of novel endocannabinoid signaling in early land plants and a future direction to elucidate its functional role.

No description available.