O hormônio tireotrófico (TSH) é o principal regulador da síntese e da secreção dos hormônios tireoidianos (HTs), os quais exercem um mecanismo de feedback negativo na hipófise reduzindo a síntese das cadeias <font face=\"Symbol\">&#946 e <font face=\"Symbol\">&#945 (CGA - Glycoprotein hormones Alpha Chain) por meio de mecanismos que envolvem modificações na transcrição de genes que codificam essas proteínas (ações genômicas). Na última década tem aumentado o número de evidências de que, em paralelo as ações genômicas clássicas, algumas ações dos HTs são desencadeadas na presença de inibidores da transcrição gênica e em curto espaço de tempo (segundos a minutos), caracterizando-se assim as ações não genômicas dos HTs. Este trabalho tem como foco avaliar a possibilidade de que os HTs regulem a expressão desses genes não genomicamente. Para tal avaliamos as alterações decorrentes do hipotiroidismo, seguido ou não do tratamento agudo com T3 em dose fisiológica ou saturante, sobre o grau de poliadenilação e a expressão do mRNA das subunidades alfa (CGA) e <font face=\"Symbol\">&#946TSH, bem como sua repercussão sobre a síntese e secreção de <font face=\"Symbol\">&#946TSH. Através da metodologia de PCR em Tempo Real observamos nos animais tireoidectomizados tratados com salina (Tx) um aumento de 10 e 4 vezes no conteúdo de mRNA do <font face=\"Symbol\">&#946TSH e CGA, respectivamente, e na razão <font face=\"Symbol\">&#946TSH/CGA quando comparado ao animal eutireoideo. A administração da dose saturante de T3 em 30 min não alterou o conteúdo do mRNA de <font face=\"Symbol\">&#946TSH e CGA, enquanto a dose fisiológica reduziu 52 e 34%, respectivamente, sem alterar a razão <font face=\"Symbol\">&#946TSH/CGA, comparando com o grupo Tx. Com o ensaio RACE-PAT, observou-se que o grupo Tx apresentou um aumento no comprimento da cauda poli-A do mRNA de <font face=\"Symbol\">&#946TSH, não havendo alterações semelhantes para o mRNA de CGA. A administração aguda de T3, apenas na dose saturante, provocou uma redução de 17% no comprimento da cauda poli-A do mRNA do <font face=\"Symbol\">&#946TSH nos animais hipotiroideos comparados com o grupo Tx. Nenhuma alteração foi observada no comprimento da cauda poli-A do mRNA de CGA, indicando um possível efeito específico do T3 sobre a poliadenilação da subunidade <font face=\"Symbol\">&#946. Através dos ensaios Western Blot / ECL, Imunohistoquímica e Histoquímica foi observado que as duas doses de T3 utilizadas promoveram um aumento de 30% no conteúdo protéico de TSH, uma redução na marcação de <font face=\"Symbol\">&#946TSH na periferia dos tireotrofos e aumento na polimerização de actina na hipófise dos animais hipotiroideos tratados, possivelmente por inibir a secreção deste hormônio. Como estes resultados foram observados em 30 min, e parte deles envolveu alterações em etapas pós-transcricionais da regulação da expressão de genes (poliadenilação), podemos inferir que o T3 esteja agindo por uma via não genômica regulando a síntese e secreção do TSH. / The thyroid-stimulating hormone (TSH) is the main regulator of the synthesis and secretion of thyroid hormones (TH), which exert a negative feedback mechanism in the pituitary by reducing the synthesis of <font face=\"Symbol\">&#946 and <font face=\"Symbol\">&#945 (CGA - Glycoprotein hormones alpha chain) chains through mechanisms that involve changes in the transcription of genes that encode these proteins (known as genomic action). However, in the last decade, an increasing body of evidence has shown that, in parallel with the classical genomic mechanisms, some TH actions might be elicited in a short period time (seconds to minutes), and in the presence of gene transcription inhibitors, which indicates that TH can also act nongenomically. In the present study we evaluate if TH could regulate some steps of the expression of <font face=\"Symbol\">&#946 TSH and CGA in a short period of time, which might provide evidence that they could act by non genomic mechanisms. For this, the expression and polyadenylation of alpha (CGA) and <font face=\"Symbol\">&#946 subunits of TSH mRNA, and TSH content, were evaluated by real time PCR and western blot, respectively, in thyroidectomized (hypothyroid) rats, 30 min after they were subjected or not to physiological or saturating doses of T3. It was observed that hyroidectomyzed animals treated with saline (Tx) presented an increase of 10 and 4 times in the content of <font face=\"Symbol\">&#946TSH and CGA mRNA, respectively, and in the <font face=\"Symbol\">&#946TSH / CGA ratio compared with control group. The saturating dose of T3 did not alter the <font face=\"Symbol\">&#946TSH and CGA mRNAs content, but the physiological dose reduced them at 52 and 34% respectively, without changing the <font face=\"Symbol\">&#946TSH / CGA ratio, compared with Tx group. The RACE-PAT assay showed that the Tx rats presented an increase in the mRNA <font face=\"Symbol\">&#946TSH poly-A tail length, whereas no change was observed to the mRNA of CGA. The acute and saturating dose of T3 caused a 17% reduction in the length of mRNA <font face=\"Symbol\">&#946TSH poly-A tail in hypothyroid animals compared with hypothyroid group. No changes were observed in the length of the poly-A tail of mRNA CGA, suggesting a specific effect of T3 on the <font face=\"Symbol\">&#946 subunit polyadenylation. Through the Western blot/ECL, histochemistry and immunohistochemistry methods we could observe that T3 (in both doses used) promoted a 30% increase in TSH protein content, a decrease in <font face=\"Symbol\">&#946TSH labeling near thyrotrophs plasma membrane and increased the actin polymerization in the pituitary of hypothyroid animals, possibly by inhibiting the secretion of this hormone. Considering that these results were observed in 30 min, and some of them involve changes in post-transcriptional regulation of gene expression (polyadenylation), we can infer that in parallel to its genomic action, T3 acts by non genomic pathways in the regulation of the TSH synthesis and secretion.

O hormônio tireotrófico (TSH) é o principal regulador da síntese e da secreção dos hormônios tireoidianos (HTs), os quais exercem um mecanismo de feedback negativo na hipófise reduzindo a síntese das cadeias beta (Tshb) e alfa (Cga) por meio de ações genômicas. Em paralelo, algumas ações dos HTs são desencadeadas na presença de inibidores da transcrição gênica e em segundos a minutos, caracterizando-se assim as ações não genômicas. O objetivo deste estudo foi avaliar as possíveis ações não genômicas do T3 sobre a expressão, processamento pós-transcricional, tradução e secreção do TSH, e a participação do cálcio e magnésio neste processo. Os resultados demonstraram que o T3, via interação com a integrina aVb3, reduz o conteúdo de mRNA de Tshb mesmo na presença de bloqueador da transcrição gênica, o comprimento da cauda poli(A), a taxa de tradução deste transcrito e a secreção por vias dependentes da integrina aVb3 e PI3K. E também aumenta a concentração intracelular de magnésio mas não altera a de cálcio. Estes dados evidenciam a existência de um mecanismo adicional e não genômico pelo qual o T3 interage com a integrina aVb3 e reduz a síntese/secreção de TSH que se soma ao já conhecido efeito de feedback negativo via controle da taxa de transcrição gênica de Tshb e Cga. / The thyrotropin (TSH) is the main regulator of thyroid hormones (HTs) synthesis and secretion, which in turn, exert a negative feedback in the pituitary gland reducing the synthesis of alpha (Cga) and beta (Tshb) TSH subunits by genomic actions. In parallel, some HTs actions are triggered in the presence of inhibitors of gene transcription and in seconds to minutes, featuring the non genomic actions of HTs. The goal of this study was to evaluate the possible non genomic actions of T3 on expression, posttranscriptional regulation, translation and secretion of TSH, as well as, the participation of calcium and magnesium on this process. The results have shown that the T3, interacts with aVb3 integrin, reduces the content of Tshb mRNA even in the presence of gene transcription inhibitor, decreases the poly(A) tail length, the translation rate of this transcript and the secretion through aVb3- and PI3K-dependent mechanisms. The T3 also increases and does not alter the intracellular concentration of magnesium and calcium, respectively. These data demonstrates the existence of an additional and non genomic mechanism by which the T3 interacts with aVb3 integrin and reduces the synthesis/secretion of TSH, in parallel to the control of Tshb and Cga gene transcription.

In vertebrate oocytes, meiotic progression is driven by the sequential translational activation of maternal messenger RNAs stored in the cytoplasm. This activation is mainly induced by the cytoplasmic elongation of their poly(A) tails, which is mediated by the cytoplasmic polyadenylation element (CPE) present in their 3’ untranslated regions (3´ UTRs). Sequential, phase-specific translation of these maternal mRNAs is required to complete the two meiotic divisions. Although the earlier polyadenylation events in prophase I and metaphase I are driven by the CPE-binding protein 1 (CPEB1), 90% of this protein is degraded by the anaphase promoting complex in the first meiotic division. The low levels of CPEB1 during interkinesis and in metaphase II raise the question of how the cytoplasmic polyadenylation required for the second meiotic division is achieved. In this work, we demonstrate that CPEB1 activates the translation of the maternal mRNA encoding CPEB4, which, in turn, recruits the cytoplasmic poly(A) polymerase GLD2 to “late” CPE-regulated mRNAs driving the transition from metaphase I to metaphase II, and, therefore, replacing CPEB1 for “late” meiosis polyadenylation.

Mon travail de thèse porte sur l’étude du rôle de l’histone acétyl-transférase MOZ et de l’histone méthyle-transférase MLL dans l’hématopoïèse. Elles contrôlent l’expression de nombreux gènes, nottament des gènes HOX, des facteurs de transcription connus pour leur rôle dans l’hématopoïèse normale et pathologique. Les deux protéines ont des gènes cibles communs tel qu'HOXA9. Ces observations nous ont conduit à rechercher une coopération fonctionnelle entre MOZ et MLL. Nous avons montré que MOZ était associée avec MLL dans les cellules souches/progénitrices humaines CD34+ afin d’activer la transcription des gènes HOXA5, HOXA7 et HOXA9. En effet, les deux protéines interagissent et sont recrutées au niveau de leur promoteur. Nous avons mis en évidence une interférence fonctionnelle entre ces deux facteurs épigénétiques, puisque MOZ est nécessaire au recrutement et à l’activité enzymatique de MLL au niveau des gènes HOXA5, HOXA7 et HOXA9 et réciproquement.Afin de caractériser le mécanisme d’action impliquant la coopération entre MOZ et MLL, nous avons recherché d’autres partenaires associés à ce duo. Nous avons identifié la Symplekin, un membre de la machinerie de polyadénylation. Nous avons mis en évidence l’interaction de la Symplekin avec MOZ et MLL dans les cellules de la lignée hématopoïétique humaine KG1. Les trois protéines sont co-recrutées sur le promoteur du gène HOXA9. Nous avons démontré le rôle ambivalent de la Symplekin. Bien qu’elle soit importante pour la polyadénylation et par conséquent pour la stabilité de l’ARN Hoxa9, la Symplekin empêche le recrutement de MOZ et de MLL au niveau du gène HOXA9, conduisant ainsi à une diminution de sa transcription. / My thesis project has consisted of the study of MOZ, and MLL. They are epigenetic regulators. MOZ and MLL activate transcription of HOX genes, which are transcription factors essential during haematopoiesis. MOZ and MLL have some target genes in common. In our study, we characterised a cooperation between MOZ and MLL in human haematopoietic stem/progenitor cells CD34+. They are both recruited onto HOX promoters. MOZ is essential for MLL recruitment, and this is reciprocal. In conclusion, we provided an example of a mechanism involving a direct cross-talk between two histone modifying enzymes.In order to dissect the mechanism of action of this complex, we decided to identify novel proteins interacting with both MOZ and MLL. A member of the RNA polyadenylation machinery has been isolated: Symplekin. We confirmed the interaction between MOZ, MLL and Symplekin in the human haematopoietic immature cell line KG1. We showed that Symplekin is co-recruited to HOXA9 promoter along with MOZ and MLL. We demonstrated the dual role of this member of the polyadenylation machinery. Indeed, besides the fact that Symplekin is important for Hoxa9 polyadenylation, thus its stability, it prevents MOZ and MLL recruitment onto HOXA9 promoter, leading to a decrease of HOXA9 transcription.Our work improved the understanding of the mechanism of action of MOZ and MLL in HOX control.

O hormônio tireotrófico (TSH) é o principal regulador da síntese e da secreção dos hormônios tireoidianos (HTs), os quais exercem um mecanismo de feedback negativo na hipófise reduzindo a síntese das cadeias <font face=\"Symbol\">&#946 e <font face=\"Symbol\">&#945 (CGA - Glycoprotein hormones Alpha Chain) por meio de mecanismos que envolvem modificações na transcrição de genes que codificam essas proteínas (ações genômicas). Na última década tem aumentado o número de evidências de que, em paralelo as ações genômicas clássicas, algumas ações dos HTs são desencadeadas na presença de inibidores da transcrição gênica e em curto espaço de tempo (segundos a minutos), caracterizando-se assim as ações não genômicas dos HTs. Este trabalho tem como foco avaliar a possibilidade de que os HTs regulem a expressão desses genes não genomicamente. Para tal avaliamos as alterações decorrentes do hipotiroidismo, seguido ou não do tratamento agudo com T3 em dose fisiológica ou saturante, sobre o grau de poliadenilação e a expressão do mRNA das subunidades alfa (CGA) e <font face=\"Symbol\">&#946TSH, bem como sua repercussão sobre a síntese e secreção de <font face=\"Symbol\">&#946TSH. Através da metodologia de PCR em Tempo Real observamos nos animais tireoidectomizados tratados com salina (Tx) um aumento de 10 e 4 vezes no conteúdo de mRNA do <font face=\"Symbol\">&#946TSH e CGA, respectivamente, e na razão <font face=\"Symbol\">&#946TSH/CGA quando comparado ao animal eutireoideo. A administração da dose saturante de T3 em 30 min não alterou o conteúdo do mRNA de <font face=\"Symbol\">&#946TSH e CGA, enquanto a dose fisiológica reduziu 52 e 34%, respectivamente, sem alterar a razão <font face=\"Symbol\">&#946TSH/CGA, comparando com o grupo Tx. Com o ensaio RACE-PAT, observou-se que o grupo Tx apresentou um aumento no comprimento da cauda poli-A do mRNA de <font face=\"Symbol\">&#946TSH, não havendo alterações semelhantes para o mRNA de CGA. A administração aguda de T3, apenas na dose saturante, provocou uma redução de 17% no comprimento da cauda poli-A do mRNA do <font face=\"Symbol\">&#946TSH nos animais hipotiroideos comparados com o grupo Tx. Nenhuma alteração foi observada no comprimento da cauda poli-A do mRNA de CGA, indicando um possível efeito específico do T3 sobre a poliadenilação da subunidade <font face=\"Symbol\">&#946. Através dos ensaios Western Blot / ECL, Imunohistoquímica e Histoquímica foi observado que as duas doses de T3 utilizadas promoveram um aumento de 30% no conteúdo protéico de TSH, uma redução na marcação de <font face=\"Symbol\">&#946TSH na periferia dos tireotrofos e aumento na polimerização de actina na hipófise dos animais hipotiroideos tratados, possivelmente por inibir a secreção deste hormônio. Como estes resultados foram observados em 30 min, e parte deles envolveu alterações em etapas pós-transcricionais da regulação da expressão de genes (poliadenilação), podemos inferir que o T3 esteja agindo por uma via não genômica regulando a síntese e secreção do TSH. / The thyroid-stimulating hormone (TSH) is the main regulator of the synthesis and secretion of thyroid hormones (TH), which exert a negative feedback mechanism in the pituitary by reducing the synthesis of <font face=\"Symbol\">&#946 and <font face=\"Symbol\">&#945 (CGA - Glycoprotein hormones alpha chain) chains through mechanisms that involve changes in the transcription of genes that encode these proteins (known as genomic action). However, in the last decade, an increasing body of evidence has shown that, in parallel with the classical genomic mechanisms, some TH actions might be elicited in a short period time (seconds to minutes), and in the presence of gene transcription inhibitors, which indicates that TH can also act nongenomically. In the present study we evaluate if TH could regulate some steps of the expression of <font face=\"Symbol\">&#946 TSH and CGA in a short period of time, which might provide evidence that they could act by non genomic mechanisms. For this, the expression and polyadenylation of alpha (CGA) and <font face=\"Symbol\">&#946 subunits of TSH mRNA, and TSH content, were evaluated by real time PCR and western blot, respectively, in thyroidectomized (hypothyroid) rats, 30 min after they were subjected or not to physiological or saturating doses of T3. It was observed that hyroidectomyzed animals treated with saline (Tx) presented an increase of 10 and 4 times in the content of <font face=\"Symbol\">&#946TSH and CGA mRNA, respectively, and in the <font face=\"Symbol\">&#946TSH / CGA ratio compared with control group. The saturating dose of T3 did not alter the <font face=\"Symbol\">&#946TSH and CGA mRNAs content, but the physiological dose reduced them at 52 and 34% respectively, without changing the <font face=\"Symbol\">&#946TSH / CGA ratio, compared with Tx group. The RACE-PAT assay showed that the Tx rats presented an increase in the mRNA <font face=\"Symbol\">&#946TSH poly-A tail length, whereas no change was observed to the mRNA of CGA. The acute and saturating dose of T3 caused a 17% reduction in the length of mRNA <font face=\"Symbol\">&#946TSH poly-A tail in hypothyroid animals compared with hypothyroid group. No changes were observed in the length of the poly-A tail of mRNA CGA, suggesting a specific effect of T3 on the <font face=\"Symbol\">&#946 subunit polyadenylation. Through the Western blot/ECL, histochemistry and immunohistochemistry methods we could observe that T3 (in both doses used) promoted a 30% increase in TSH protein content, a decrease in <font face=\"Symbol\">&#946TSH labeling near thyrotrophs plasma membrane and increased the actin polymerization in the pituitary of hypothyroid animals, possibly by inhibiting the secretion of this hormone. Considering that these results were observed in 30 min, and some of them involve changes in post-transcriptional regulation of gene expression (polyadenylation), we can infer that in parallel to its genomic action, T3 acts by non genomic pathways in the regulation of the TSH synthesis and secretion.

The life-cycle of RNA is determined by several processing steps, which allow the cell to export and translate a coding transcript. The cell has developed an astonishingly complex mechanism to ensure the integrity of RNA processing steps. The quality control mechanism of RNA balances the biosynthesis and degradation of various transcripts, adding another layer of gene regulation to the complex system of gene expression. The exosome is a central piece of the RNA quality control mechanism as it degrades many of the aberrant or non-functional RNAs in the nucleus and the cytoplasm. This project characterizes and highlight a response to mutation of components from the RNA quality control mechanism in Saccharomyces cerevisiae. These perturbations include functional components of the exosome (Csl4 and Dis3), a cofactor of the nuclear exosome (Rrp6), an essential protein for pre-rRNA processing (Enp1) and a component of RNA export machinery (Srm1). Here, I present bioinformatics approaches to characterize the cellular response at a level of transcript expression and polyadenylation size. The stress response embedded in the gene expression profile is highly similar between the mutants. This work suggests a generic response to a failure in different components of the RNA quality control machinery. / Le cycle de vie des ARN est déterminé par différentes étapes permettant à la cellule d’exporter et de traduire un transcrit codant. La cellule a développé un mécanisme incroyablement complexe pour s’assurer de l’intégrité des étapes de maturation de l’ARN. Le mécanisme de contrôle de qualité balance la biosynthèse et la dégradation de différents transcrits, ce qui ajout un niveau de régulation au système de l’expression génique. L’exosome est une pièce centrale du mécanisme de contrôle de qualité de l’ARN alors qu’elle dégrade une grande partie des transcrits aberrants ou non-fonctionnels dans le noyau et le cytoplasme. Ce projet caractérise et souligne la réponse cellulaire à la suite de la mutation de composantes du mécanisme de contrôle de qualité de l’ARN chez Saccharomyces cerevisiae. Ces perturbations comportent des composantes fonctionnelles du complexe de l’exosome (Csl4 et Dis3), un cofacteur de l’exosome nucléaire (Rrp6), une protéine essentielle pour la maturation des pré-ARNr (Enp1) et une composante de la machinerie d’export de l’ARN (Srm1). Ici, je présente des approches bio-informatiques pour caractériser la réponse cellulaire au niveau de l’expression des transcrits et de la taille des segments polyadénylés. La réponse au stress cellulaire intégré dans le profil d’expression du génome est très similaire entre les mutants. Ce travail suggère une réponse générique à la suite de la perturbation de différentes composantes du mécanisme de contrôle de qualité de l’ARN.

Polyadenylation signals in yeast are not very well defined and are believed to be largely degenerate. Here, we present a computational and experimental genome-wide analysis of polyadenylation signals in Schizosaccharomyces pombe (S. pombe), identifying the canonical AATAAA motif as the most frequent and functional signal. RNA-Seq data from cells grown under various physiological conditions were used to map 3’UTRs, which classify as commonly heterogenic. We have shown that many genes have alternative 3’UTRs. Our results are summarised and can be accessed in a user-friendly online database Pomb(A). It has been shown that convergent genes require trans elements, like Cohesin, for efficient transcription termination. We demonstrate that convergent genes lacking Cohesin are generally associated with longer overlapping transcripts. Furthermore, we analysed ChIP-chip data of Rad21 and Mis4 as well as other Cohesin and loading complex subunits and show that regions of Rad21/Mis4 co-localisation are generally associated with highly transcribed genes. They are also cohesive, while sites with Rad21 only are less cohesive. Rad21/Mis4 co-localisation sites are in close proximity to annotated origins of replication, suggesting that cohesive sites may facilitate replication. microRNAs (miRNAs) are well studies in higher eukaryotes and participate on post-transcriptional gene silencing by degrading target mRNA or blocking translation. It is believed that miRNAs do not exist in yeast. We reanalyzed miRNA presence in yeast using recently available small RNA data sets. Potential miRNA genes and targets in S. pombe were computationally predicted based on the described alternative 3’UTR data and further experimentally tested. Dicer is an enzyme, which recognizes long dsRNA substrates and cleaves them into siRNA e↵ector molecules, essential for gene silencing. Dicer has been thought to be a purely cytoplasmic protein. However, we employed ChIP-Seq and dsRNA RNA-Seq data to show that Dicer localises in the nucleus of mammalian cells and associates with the chromatin on numerous loci. Furthermore, we present evidence that Dicer processes long dsRNA into siRNA in the nucleus and the lack of Dicer causes the accumulation of long dsRNA. This consequently induces the interferon response pathway, which ultimately leads to apoptosis and cell death.

<p>High-risk type human papillomaviruses (HPVs) are associated with cancer. HPVs are strictly epitheliotropic and infect basal cell layers, establishing a life cycle strongly linked to the differentiation stage of the infected cells. The viral capsid late genes, L2 and L1, are only expressed in terminally differentiated epithelium. Late gene expression involves regulation of most gene processing events including transcription, splicing, polyadenylation, mRNA stability and translation. </p><p>Both L2 and L1 have elements present in the open reading frames (ORFs) negatively affecting mRNA levels and translation. The negative elements in L1 were mapped to the first 514 nucleotides, with the strongest inhibitory effect located in the first 129 nucleotides. The negative elements in the L2 sequence were concentrated in two locations on the gene. Both genes were mutated by changing the nucleotide sequence while retaining the amino acid sequence. Mutating the first 514 nucleotides in L1 deactivated the negative elements while the entire L2 gene had to be mutated to achieve the same result. The L2 protein was found to localise the L1 protein into a punctuated pattern in the nucleus.</p><p>In the HPV-16 genome the negative elements reside in regions important for regulation of polyadenylation and splicing, critical for late gene expression. By exchanging parts of the L2 gene in subgenomic constructs with the corresponding mutant sequence we show that certain features of the L2 elements direct splicing to the L1 splice acceptor, and also regulate the efficiency of the early polyadenylation site. Cumulative binding of hnRNP H to the L2 mRNA gradually increased polyadenylation efficiency. Most interestingly, hnRNP H levels were downregulated in more differentiated epithelial cells. </p><p>Elucidation of how expression of the immunogenic late proteins is regulated would be greatly beneficial in prevention and treatment of HPV infection and thereby cancer.</p>

High-risk type human papillomaviruses (HPVs) are associated with cancer. HPVs are strictly epitheliotropic and infect basal cell layers, establishing a life cycle strongly linked to the differentiation stage of the infected cells. The viral capsid late genes, L2 and L1, are only expressed in terminally differentiated epithelium. Late gene expression involves regulation of most gene processing events including transcription, splicing, polyadenylation, mRNA stability and translation. Both L2 and L1 have elements present in the open reading frames (ORFs) negatively affecting mRNA levels and translation. The negative elements in L1 were mapped to the first 514 nucleotides, with the strongest inhibitory effect located in the first 129 nucleotides. The negative elements in the L2 sequence were concentrated in two locations on the gene. Both genes were mutated by changing the nucleotide sequence while retaining the amino acid sequence. Mutating the first 514 nucleotides in L1 deactivated the negative elements while the entire L2 gene had to be mutated to achieve the same result. The L2 protein was found to localise the L1 protein into a punctuated pattern in the nucleus. In the HPV-16 genome the negative elements reside in regions important for regulation of polyadenylation and splicing, critical for late gene expression. By exchanging parts of the L2 gene in subgenomic constructs with the corresponding mutant sequence we show that certain features of the L2 elements direct splicing to the L1 splice acceptor, and also regulate the efficiency of the early polyadenylation site. Cumulative binding of hnRNP H to the L2 mRNA gradually increased polyadenylation efficiency. Most interestingly, hnRNP H levels were downregulated in more differentiated epithelial cells. Elucidation of how expression of the immunogenic late proteins is regulated would be greatly beneficial in prevention and treatment of HPV infection and thereby cancer.

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;