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Short noncoding RNAs play roles in regulating nearly every biological process, in nearly every organism, yet the exact function and importance of these molecules remains a subject of some debate. In order to gain a better understanding of the contexts in which these regulators have evolved, I have undertaken a variety of approaches to study the evolutionary history of the components that make up these pathways, in the form of two main research efforts. In the first chapter, I have used a combination of population genetics and molecular evolution techniques to show that proteins involved in the piRNA pathway are rapidly evolving, and that different components of the pathway seem to be evolving rapidly on different timescales. These rapidly evolving piRNA pathway proteins can be loosely separated into two groups. The first group appears to evolve quickly at the species level, perhaps in response to transposons that invade across species lines, while the second group appears to evolve quickly at the level of individual populations, perhaps in response to transposons that are paternally present yet novel to the maternal genome. In the second chapter of my research, I have used molecular evolution techniques and carefully devised controls to show that the binding sites of well-conserved miRNAs are among the most slowly changing short motifs in the genome, consistent with a conserved function for these short RNAs in regulatory pathways that are ancient and extremely slow to change. I have additionally discovered a major flaw in an existing approach to motif turnover calculations, which may lead to systematic biases in the published literature toward the false inference of increased regulatory complexity over time. I have implemented a revised approach to motif turnover that addresses this flaw.

The formation of the mRNP complex is a critical component of translational regulation and mRNA decay. Both the 5 ' and 3 ' UTRs of CSF-1 mRNA are involved in post-transcriptional regulation. In CSF-1 mRNA, a small hairpin loop structure is predicted to form at the extreme 5 ' end (2-21 nt) of the 5 ' UTR. Nucleolin binds the hairpin loop structure in the 5 ' UTR of CSF-1 mRNA and enhances translation, while removal of this hairpin loop nucleolin binding element dramatically represses translation. Thus in CSF-1 mRNA, the hairpin loop nucleolin binding element is critical for translational regulation. In addition, nucleolin interacts with the 3 ' UTR of CSF-1 mRNA and facilitates the miRISC formation which results in poly (A) tail shortening. The overexpression of nucleolin increases the association of CSF-1 mRNA containing short poly (A)(n), <= 26, with polyribosomes. Nucleolin both forms an mRNP complex with the eIF4G and CSF-1 mRNA, and is co-localized with the eIF4G in the cytoplasm further supporting nucleolin's role in translational regulation. The distinct foci formation of nucleolin in the cytoplasm of ovarian and breast cancer cells implicates the translational promoting role of nucleolin in these cancers.

abstract: Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known. MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues. I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

<p>Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. </p><p>A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised <i>in vitro </i>and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an <i>in vitro </i>stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation.</p><p>In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.</p>

RNA sequences and higher-order structures in the 5’ and 3’ untranslated regions (UTRs) of positive-strand RNA viruses are known to function as cis-acting elements for translation, replication, and transcription. In coronaviruses, these are best characterized in the group 2a bovine coronavirus (BCoV) and mouse hepatitis virus (MHV), yet their precise mechanistic features are largely undefined. Here, we use a reverse genetics system in MHV to exploit the ~30% nt sequence divergence between BCoV and MHV to establish structure/function relationships of 5’ UTR cis-replication elements. It had been previously shown that a precise replacement of the 391-nt MHV 3’ UTR with the 288-nt BCoV 3’ UTR yields wt-like MHV. Our attempts to replace the 209-nt MHV 5’ UTR with the 210-nt BCoV 5’ UTR, however, yielded a non-viable chimera. Therefore, a systematic analysis of individual 5’-terminal structures was made to identify compatible elements. By placing each of four putative cis-acting domains from the BCoV 5’ UTR into the MHV genome, we learned that (i) stem-loops (SLs) I & II and SLIII are functionally compatible, (ii) SLIV is compatible if it spans parts of the 5’ UTR and the nonstructural protein 1 (nsp1) cistron, thus identifying this part of ORF 1 as a component of the cis-replication signal, (iii) a relatively unstructured 32-nt region mapping between SLIII and SLIV defines a novel virus species-specific cis-replication element, (iv) spontaneous suppressor mutations within MHV SLI and nsp1 cistron compensated for growth defects arising from the BCoV 32-nt element in the MHV genome, (v) cross talk between the 32-nt element, SLI, and the nsp1 cistron appears essential for virus replication, (vi) the BCoV 5’ UTR and nsp1 cistron function together in the MHV genome to generate a wt-like MHV phenotype, and (vii) a functional 5’ UTR-nsp1 domain in group 2a coronaviruses cannot be substituted by the corresponding genomic element from the group 2b SARS-CoV. We postulate that the interaction between the 5’ UTR and nsp1 cistron (or possibly nsp1 protein) functions as a molecular switch between genome translation and ignition of negative-strand RNA synthesis.

Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised in vitro and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an in vitro stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation. In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.

Orientador: Sérgio Roberto Peres Line / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T18:59:49Z (GMT). No. of bitstreams: 1 Urioste_EduardoArcanjo_M.pdf: 1274507 bytes, checksum: 0f7136d4dabaf0e810ad2bdf1b2ee815 (MD5) Previous issue date: 2013 / Resumo: Estima-se que cada indivíduo carregue cerca de 120 a 430 variantes raras em regiões UTRs (Abecasis et al, 2012). Apesar da tolerância a variação na região 5' UTR, a patofisiologia de várias doenças está ligada a mutações na mesma (Cazzola & Skoda, 2000; Reynolds, 2002; Chatterjee & Pal, 2009; Wethmar et al 2010), sendo necessário o entendimento a determinação dos mecanismos regulatórios. O objetivo deste trabalho é descobrir assinaturas genéticas encontradas no contexto genômico de mutações pontuais de região 5' UTR que permitam prever o impacto funcional de outras variações pontuais na mesma região. As mutações, causadora de doença, foram selecionadas do banco de dados do Human Gene Mutation Database (HGMD) (Stenson et al, 2008); e os polimorfismos, de impacto funcional desconhecido, foram obtidos no banco de dados NHLBI Grand Opportunity Exome Sequencing Project (ESP), sendo originados do trabalho de Tenessen et al (2012). No total foram utilizadas 235 mutações e 21.542 polimorfismos. Para as variações foram calculados parâmetros de variação da estabilidade da estrutura secundária do contexto das variações (??Gfolding), presença de sítios de ligação de fatores de transcrição (JASPAR), tipo de variação (transição/transversão, tipoV), distância do início da sequência codificante (DiSC), distância do início de transcrição (DiTr) e conservação filogenética por distância de Levenshtein do contexto (Lev). A estatística foi calculada pelos testes de Wilcoxon e Binomial. A partir destes foram gerados modelos de regressão logísticos analisados através de curva ROC. Os parâmetros ??Gfolding máximo, tipoV, DiSC, e Lev permitiram a distinção significativa (? = 0,05) entres os polimorfismos e as mutações permitindo modelos explicativos, mas incompletos (área da Curva ROC 0, 772). ??Gfolding max. indicou uma relação entre as mutações e entre estruturas secundárias mais estáveis geradas pelas mesmas. Os parâmetros Lev e tipoV sugerem a origem das mutações como resultantes de hotspots. O parâmetro DiSC indicou regiões com provável funcionalidade. Apesar de não ter sido possível estabelecer relação causal entre os parâmetros e o impacto funcional das variações, encontrou-se correlações importantes / Abstract: It is estimated that each individual carries about 120 to 430 rare variante in the UTR regions (Abecasis et al, 2012). Despite the increased tolerance towards variations in 5' UTR region, the patho-phisiology of several diseases is linked to its mutations (Cazzola & Skoda, 2000; Reynolds, 2002; Chatterjee & Pal, 2009; Wethmar et al 2010). Therefore it is necessary the understanding and the determination of the regulatory elements. The objective of this study is the discovery of genetic signatures found in the genomic context of disease causing point mutations in 5' UTR, thus allowing the prediction of the functional impact of other point variations in the same region. The disease causing mutations were selected from Human Gene Mutation Database (HGMD) (Stenson et al, 2008). The polymorphisms of unknown functional impact were obtained from the NHLBI Grand Opportunity Exome Sequencing Project (ESP), originated from the work of Tenessen et al (2012). A total of 235 mutations and 21,542 polymorphisms were used. For each variation, parameters related with the differences of the variation's context folding stability (??Gfolding), presence of transcription factor binding sites (JASPAR), type of variation (transition/transversion, tipoV), distance from coding sequence start (DiSC), distance from transcription start site (DiTr) and phylogenetic conservations by distance of Levenshtein from wild type to variant context (Lev). The statistical test was done by Wilcoxon and Binomial. Logistical regressions models were generated from the parameters and its performance was evaluated by a ROC curve. The parameters maximal ??Gfolding, tipoV, logarithm of DiSC and Lev allowed a significant distinction (? = 0,05) between the groups, generating models of reasonable explanation but incomplete (area under the ROC curve 0,772). Maximal ??Gfolding showed a relationship between mutations and stable secondary structures generated by them. Lev and tipoV suggested the origin of the mutation from hotspots. The DiSC parameter identified regions with possible functionality. While it was not possible to establish any clear causal relationship between the parameters and the functional impact of the variations, important correlations were found / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental